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The permeability of Drosophila melanogaster embryos Watson, Catherine E. 1990

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T h e P e r m e a b i l i t y of Drosophila  melanogaster  Embryos  by  Catherine E. Watson B. Sc. The University of British Columbia,  1987  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Biochemistry  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA March 1990 © Catherine E. Watson,  1990.  In presenting  this thesis in partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may department or by  his or her  representatives.  be granted by the head of  It is understood that copying or  publication of this thesis for financial gain shall not be allowed without my permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  my  written  ii ABSTRACT Drosophila  now  are used extensively for genetic, developmental and  molecular  transformation  biology of these  research. organisms  At  present,  can only  germline  be achieved  by  microinjection of P-element vectors into the pole cells of young embryos.  The technique  of microinjection however, requires a  delicate touch and is quite laborious. a rapid and simple technique Electroporation, like  Therefore, the development of  was investigated.  microinjection, is a physical means of  introducing D N A into a cell and is therefore potentially applicable to all cell types.  Electroporation involves the use of an electrical  current to create pores in the membrane of a cell. such as D N A may enter a cell via these pores. quick, reproducible, and efficient  method  Macromolecules,  Electroporation is a  for transforming  Drosophila  Through studies of the survival and permeability of melanogaster  embryos  discovered  that although  exposed  to electrical  cells.  currents, i t was  the survival of the embryos decreased  steadily as field strength increased, the embryos did not become permeable to a water soluble dye unless a pulse of 10 kV/cm was applied. dye  Few embryos survived this extreme voltage required for  uptake.  Drosophila  Attempts  to introduce  DNA  embryos utilizing this technique  transformants. protective coatings  These  results  suggested  of the dechorionated  into  dechorionated  however, produced no that  the  remaining  embryo were obstructing  efficient pore formation, thus preventing D N A  penetration.  In view of these results, methods to eliminate the wax layer, present  between the chorion and vitelline membrane of laid eggs,  iii were examined.  Wax  extraction and melting  removal by detergent by heating  produce a satisfactory procedure.  solubilization, solvent  were investigated, yet did not  iv T A B L E OF CONTENTS Page  Abstract Table of Contents List of Tables List of Figures Acknowledgement Dedication List of Abbreviations I. Introduction A. Drosophila as an Organism for Scientific Research B. Drosophila Transformation 1. The germline cells 2. Microinjection 3. P-elements C Electroporation D. Xanthine Dehydrogenase 1. The rosy locus 2. Purine selection E The Drosophila Eggshell 1. The composition of the eggshell 2. Embryo permeability F. Scope of this Thesis II. Materials and Methods A. Materials B. Buffers and Solutions C D N A Amplification and Purification 1. Bacterial strains 2. Vectors 3. Bacterial transformation 4. Plasmid isolation a. M i n i preps b. Large scale plasmid isolation D. Southern Analysis of Drosophila Genomic D N A 1. Rapid phenol extraction of genomic D N A 2. Enzymatic digestion of genomic D N A 3. Southern Analysis a. Southern transfer b. Preparation of nick-translated probe c. Hybridization of filters d. Washing of filters  ii iv vii viii x xi xii 1 1 1 3 4 6 10 10 1 1 12 12 14 16 17 17 19 19 19 19 20 20 21 22 22 23 24 24 24 25 25  V  Page  E  Drosophila Embryos 1. Drosophila melanogaster  strains Egg collection and dechorionation Assessment of embryo survival Assessment of embryo permeability Tape-mounted embryos Non-mounted embryos a. Detergent-treated embryos b. Solvent-treated embryos c. Heat-treated embryos 7. Electroporation of embryos F. Purine Selection 1. Purine titre 2. Purine selection of putative transformants I I I . Results and Discussion A. Effect of the Exposure of Drosophila Embryos to an Electrical Current 1. Survival of embryos exposed to increasing field strengths 2. Permeability of embryos exposed to increasin field strengths 3. Survival of embryos exposed to increasing capacitance 4. Permeability of embryos exposed to increasing capacitance 5. Survival of embryos exposed to an electrical current in the presence of D N A 6. Discussion B. Electroporation of Drosophila Embryos 1. Survival of electroporated embryos from egg to adult 2. Phenotypic selection of putative transformants 3. Chemical analysis of putative transformants a. Purine selection titre b. Purine selection of putative transformants 4. Molecular analysis of putative transformants 5. Discussion C The Survival and Permeability of Dechorionated Embryos 1. Variation i n the survival of dechorionated embryos 2. The permeability of dechorionated embryos 3. Discussion 2. 3. 4. 5. 6.  26 26 26 27 27 27 28 28 28 29 29 29 29 30  3 1 3 1 3 1 31 35 35 35 40 40 42 42 42 42 45 45 50 50 50 52  vi D.  Permeability of Embryos Treated with TX-100 1. Survival of embryos treated with increased concentrations of TX-100 2. Permeability of embryos treated with increased concentrations of TX-100 3. Discussion E Permeability of Embryos Treated with Solvents 1. Survival of heptane-treated embryos incubated in different buffers 2. Permeability of heptane-treated embryos incubated in different buffers 3. Survival of embryos treated with different solvents * 4. Permeability of embryos treated with different solvents 5. Discussion F. Effect of Temperature on Embryo Permeability 1. Survival of embryos at increasing temperatures 2. Permeability of embryos at increasing temperatures 3. Discussion G Conclusions V I . References  Page  53 53 55 55 56 56 57 57 57 61 63 63 63 65 66 68  vii LIST OF  I.  TABLES  Analysis of the electroporation of r pCarnegie 20 and p7t25.1  Page 5 0 6  /r  5 0 6  embryos with 4 3  viii LIST O F FIGURES Page  1.  Embryonic development of Drosophila  2  2.  Pore formation and D N A integration  during  electroporation  7  3.  The main layers of the Drosophila  4.  The effect of increasing field strength on embryo  eggshell  survival 5.  13  32  The effect of increasing field strength on embryo survival and permeability to the dye Methyl Red  6.  33  The effect of increasing capacitance on embryo survival and permeability to the dye Nile Blue  7.  34  The effect of varying capacitance on the field strength survival curve  8.  36  The effect of the presence of D N A during pulsing on the survival of embryos exposed to an electrical  9.  current  37  The effect of the presence of D N A during pulsing on the development of embryo to adult  41  10.  Purine selection titre  44  11.  Southern analysis of putative transformants  46  12.  rosy  47  13.  Variability i n the survival of dechorionated  locus and pCarnegie 20 vector  embryos  51  IX  Page  14.  The effect of TX-100 concentration on embryos survival and permeability to the dye Nile Blue  15.  Survival and permeability of heptane treated embryos  16.  54  58  The effect of different solvents on embryo survival and permeability to the dye Nile Blue  17.  The effect of different solvents on embryo survival and permeability to Blue Dextran  18.  59  60  The effect of increasing temperature on embryo survival and permeability to the dye Nile Blue  64  X ACKNOWLEDGEMENT  I wish to extend my  sincere gratitude to Dr. G.M.  the opportunity and support to carry out this work. thank Dr. Ian Gillam for his helpful suggestions.  Tener for giving  I would also like to To the newly knighted  Dr. Nina Seto, thanks for all the discussions and encouragement. special thanks to those who To  me  And  a  dared to read this thesis in its early forms.  Hiron Poon, I hereby bequeath  my  bench space, and  letting you sit at your desk half the time."  "sorry for not  xi  T o those  who  haw  come into m y  and e s p e c i a l l y /or those w h o  tejt.  tije,  xii LIST O F  ABBREVIATIONS  A  adenosine  A260  absorbance at 260 nm  A 280  absorbance at 280 nm  Amp  ampicillin  BIM  basic incubation media  bp  base pair(s)  BSA  bovine  C  cytidine  cm  centimetres  cpm  counts per minute  dATP  deoxyadenosine  dCTP  deoxycytidine  dGTP  deoxyguanosine  dH20  distilled  DNA  deoxyribonucleic  D N A Pol I  DNA  DNase  serum albumin  triphosphate triphosphate triphosphate  water acid  polymerase I  deoxyribonuclease  DR  Drosophila  DTT  dithiothreitol  dTTP  deoxythymidine  EDTA  ethylenediaminetetraacetic  EtBr  ethidium  g  grams  G  guanosine  HEPES  N-2-hydroxyethylpiperazine-N-2-ethanesulfonic  hr(s)  hour(s)  Ringer's solution  triphosphate acid  bromide  acid  Xlll  IAA  isoamyl alcohol  K  1000 revolutions per minute  kb  kilobase(s)  kD  kilodalton(s)  kV  kilovolt(s)  L  litre(s)  M  molar  mA  milliampere(s)  uCi  micro curie(s)  u.Fd  microfaraday(s)  rig  microgram(s)  mg  milligram(s)  min  minute(s)  Hi  microlitre(s)  ml  millilitre(s)  mM  millimolar  u.m  micrometre(s)  msec  millisecond(s)  MW  molecular weight  ng  nanogram(s)  nm  nanometre(s)  PBSal  phosphate buffered saline  PBSuc  phosphate buffered sucrose  PBSuc TN  PBSuc with 0.05% TX-100 and 0.01% Nile Blue  PBSuc TX  PBSuc with 0.05% TX-100  r  506/ 506 r  Drosophila rosy  (96,494 coulombs)  melanogaster  strain homozygous for  mutation number 506  xiv RNase  ribonuclease  SDS  sodium dodecyl sulphate  SSC  standard  x  time  TB  terrific broth  TBE  tris-borate-EDTA  Tris  tris(hydroxymethyl)amino  tRNA  transfer  TX-100  triton  V  volts  v/v  volume per volume  wt  homozygous  w/v  weight per volume  XDH  xanthine  dehydrogenase  enzyme  Xdh  xanthine  dehydrogenase  gene  *C  degrees Celsius  saline citrate  constant  methane  RNA X-100  wild-type  1 I. A.  Introduction Drosophila  One  an  Organism for  of the most  Drosophila  which  as  -  widely  studied  Scientific  organisms  Research  i n science is  the fruit fly. Its usefulness stems from the ease i n  Drosophila  stocks  can be obtained  and maintained.  In  addition, their relatively short life cycle and ease i n mating, as well as their small genome and relatively few chromosomes make them an ideal organism for genetic studies.  As a result, a vast amount of  information on these insects has been accumulated.  They have been  used extensively for genetic and developmental studies and now in the growing field of molecular biology. Studying  the expression  interest to many scientists.  and regulation of a gene is of great Examination of the effects of altering the  nucleotide sequence of a gene or its flanking region on the in and in vitro  vivo  expression is routinely performed in many laboratories.  One problem commonly encountered however, is reintroducing the mutated  gene  back  into  Unfortunately, Drosophila  the genome  of some  organisms.  is a case in point.  B.  Drosophila  Transformation  1.  The germline  cells  Cloned D N A  must be integrated into the germline  stable transformation of a multicellular organism such as  to obtain Drosophila  melanogaster.  The  Drosophila  embryo undergoes a number of synchronous  nuclear divisions without stages  of embryonic  subsequent cell division during  development  (figure  1).  the early  Shortly  after  2  Figure  1.  Embryonic  Development  of  Drosophila  Schematic diagrams of 16 stages of Drosophila embryogenesis. The arrows trace the appearance and fate of the pole cells. (Wieschaus and N u s s l e i n - V o l h a r d , 1986). Transformation must be achieved at or prior to stage 4.  3 fertilization,several  nuclei  migrate  to the posterior end  of the  embryo, where they pinch off from the others, and form the pole cells.  Subsequently,  these  interior of the embryo Consequently, introduce  cells  where they  are directed towards the  form  the gonads of the f l y .  to produce a line of stable transformants, one must  the exogenous gene into the pole cells prior  internalization. which  pole  to their  This results in a restricted window of time during  transformation  'transformation  may  window'  occur. is  At  room  approximately  temperature, 90  minutes  this post  fertilization. 2.  Microinjection At present, the only method available to introduce D N A  pole cells of Drosophila  is by microinjection.  into the  In this procedure, the  preparation of the embryos is very important.  The embryos are  collected  formation.  and  dechorionated  prior  to pole  cell  embryos are mounted parallel to one another on double-sided  The tape  with their posterior ends over the edge of the tape, and desiccated slightly. will  The extent of desiccation is critical since inadequate drying  cause  cytoplasmic  leakage  upon  injection,  while  excessive  desiccation causes the embryo to shrivel up (Rubin and Spradling, 1982).  Both  therefore  states cause embryonic development to cease and are  unacceptable.  After optimal desiccation has been achieved, the embryos are covered  with halocarbon  o i l (to prevent  injected using a small needle (<1 mm the needle  further water loss), and  in diameter).  is used to pierce the embryo  The sharp tip of  and the D N A  solution  4  expelled (1-5% of the volume of the embryo) into the posterior pole of the embryo. chamber  The embryos are allowed to develop  and the hatched  larvae  are recovered  in a humid  and placed  on  standard food (Spradling and Rubin, 1982). The  microinjection procedure is most often done at reduced  temperature (such as 18 °C) to slow down embryonic development, thereby  extending  occur.  Also, the microinjections are performed i n high humidity to  reduce excess  the period of time i n which transformation can  desiccation of the embryo.  Microinjection however,  requires great patience, considerable manual dexterity and is quite labour intensive.  Transformation  results achieved  can vary greatly  depending primarily on the expertise of the injector. 3.  P-elements The  vectors used for transformation of Drosophila  from P-elements.  P-elements belong  are derived Drosophila  to a family of  transposable elements, that are heterogeneous i n length (0.5-2.9 kb), but homologous in sequence (O'Hare and Rubin, 1983). (2.9 kb) P-element possesses three internal open reading presumably  a transposase  The intact  perfect 31 bp terminal repeats, and frames (encoding  repressor).  a transposase, and  The smaller  P-elements  appear to have arisen from internal deletions of the larger element. These non-autonomous P-elements still retain their terminal repeats, and  are therefore able to transpose, however they  length  and  are  unable  to  produce  are shorter in their  own  transposase.transposition defective ( O'Hare and Rubin, 1983). The  introduction of P-elements  into  a genetic  background  5  lacking them (termed a M-cytotype), by microinjection (Rubin and or by genetic means (Kidwell et al , 1977), produces  Spradling, 1982)  a condition known as hybrid dysgenesis. such  traits  as, sterility,  mutations,  reversion  male recombination, of  mutations,  arrangements and non-disjunction This  This state is defined by visible  and  (Bregliano  chromosomal reand K i d w e l l , 1983).  phenomenon is due to transposon jumping  When stable germline transformation  and lethal  i n the genome.  is desired, a non-transposable  P-element is used. In germline transformation cloned  the gene of interest is  into a defective P-element (containing a selectable marker i f  required). helper  of Drosophila,  Since the element cannot induce its own transposition, a  P-element is required  required for transformation. transposable eliminating element.  P-element  to produce the transposase which is Alternatively, strains containing a non-  can be  the requirement  used  as  recipients,  thereby  of the co-injection of the helper  This increases the frequency of transformation  since only  the P-element construct must enter the pole cell. Analysis  of P-element  integration into random Spradling, 1982). been proposed  transformants  revealed  transposon  sites throughout the genome (Rubin and  Although a 8 bp target sequence ( G G C C A G A C ) has as the target for P-element insertion (O'Hare and  Rubin, 1983), it is not a stringent requirement.  The transposed D N A  shows little sign of deletions or rearrangement (Spradling and Rubin, 1982  and 1983). Although  microinjection is a relatively  efficient  method of  6 giving results ranging from < 1 to 3% of  transformation of Drosophila,  injected embryos (Scholnich et al , 1983; Wakimoto et al , 1986), the method is extremely  tedious.  Hence, a new method which is quick  and  simple, would be preferred.  C.  Electroporation  In  1982, Neumann  et al.  Electroporation has that potential.  demonstrated  that D N A  could  be  introduced into cells by applying an electrical current across a cell suspension  containing DNA.  electroporation technique  (or electroinjection).  is quite simple  Zimmermann electrical  This process  et al.  current  has since been termed  The  and stems from  (for review is applied  theory  observations 1982).  see Zimmermann,  to a suspension  electrical potential (V) is set up across  behind  this  made by When an  of membranes,  the membrane.  an  If this  potential exceeds the inherent electrical potential of the membrane (V  m a x  ),  reversible membrane breakdown w i l l  occur  (Zimmermann,  1982). Vmax =1.5 E r cos 6  (Equation 1)  0  where  V  m a  E  0  x = electric potential = field strength (V/cm)  r = cell radius cos 6 = angle between the membrane and the field direction The  lipid  molecules  become momentarily  disorganized  and  form  holes or pores in the bilayer upon reorganization (figure 2). The phospholipids continue to move, closing the pore and re-establishing the integrity of the membrane.  If the electrical potential set up  7  Figure 2.  Pore Formation and D N A Integration D u r i n g Electroporation  When a cell is subjected to an electrical field (E), an electrical potential is established across its membrane. If the potential exceeds the critical potential of the membrane, the lipids become disorganized and upon reorganization form pores. It is through these pores that the D N A can possibly enter and become integrated into the genome of the cell. The pores in the membrane gradually re-seal, re-establishing the integrity of the membrane.  8 across  the  membrane  exceeds  a  critical  value,  the  membrane  undergoes irreversible breakdown resulting in cell death. DNA  can  enter the cells through these transient pores and  into the genome (Toneguzzo et al.,  incorporated  1988).  aspect of this mass microinjection, is that the DNA pinocytosis  or endocytosis and  therefore  A positive  does not enter via  is not  subjected  to  many degradative enzymes in the lysosomal vesicles (Toneguzzo al,  1986).  be  the case for chemical transformation  As a result, the transferred DNA  be  et  procedures (Potter, 1988). integration is  If multiple inserts occur they do so at distinct loci, not as  tandem arrays. may  the  is rarely damaged, as can  In addition, results seem to indicate that the D N A random.  be  When co-integration of two  the case with  mammalian  cells  P-element transformation),  typically  gives  electroporation  between 23-77%  cells as cointegrates (Toneguzzo et al., Electroporation has  markers is desired  of  (as of  transformed  1988).  been used successfully for transformation  of  a wide variety of cell types including cultured cell lines, mammalian primary  cell  cultures, mammalian  intercellular vesicles, dicot and and  embryonic  The  cells, isolated  monocot plant cells, trypanosomes,  a variety of bacteria (for review  1988).  stem  see  Andreason and  Evans,  universal applicability of electroporation stems from the  fact that it is a physical microinjection, and  means of D N A  therefore does not rely on  introduction  - a mass  the unique properties  of the cell like other procedures do. Unfortunately, and  to  optimize  transfer efficiency, many  biological parameters must be  physical  investigated for each cell type.  9 The  two  most important  duration.  parameters are field  strength and  pulse  Their optimization w i l l determine the number, size  length of pore opening and macromolecules.  The  hence the permeability of the cell to  importance of field strength in the reversible  membrane breakdown required for pore formation above (see Equation increases in field  and  1).  Once V  has  m a x  been  was  mentioned  exceeded, further  strength cause additional pore formation  wider surface area of the cell (related to cos 0).  over a  The number and size  of the pores increase until the applied field strength creates such a great  potential across  breakdown The  the  membrane  that irreversible  membrane  occurs.  voltage stored in a capacitor decreases exponentially when  the capacitor is discharged. to decrease to 1/e  The  time required for the peak voltage  is called the time constant  compare pulse lengths.  (x) and is used to  T is dependent on two variables: the size of  the capacitor that is discharged (larger capacitors require more time to  release their charge) and  the resistance of the media through  which the electricity is discharged.  The  resistance in turn depends  on the ionic strength of the solution (higher ionic strength results in lower resistance and  hence a shorter T) and cuvette geometry.  An  increase in x results in the pores remaining open longer. The  disadvantage  that must be  of electroporation is the  examined  initially  to optimize  many  parameters  transfection.  Other  parameters which must be optimized include: DNA  concentration, topology and method of preparation  cell concentration and  growth conditions  10  pulse wave shape buffer  composition  temperature incubation  time (before and after the pulse )  (Potter, 1988). The  preliminary  work can be extensive and determining the ideal  conditions labour  intensive;  however once perfected,  the procedure  is rapid and simple.  D.  Xanthine  1.  The rosy The  rosy  Dehydrogenase  locus locus of Drosophila  melanogaster  is located on the  right arm of chromosome 3, at position 87 DE. This locus encodes the enzyme Xanthine Dehydrogenase (XDH). (rosy  -  Flies lacking this protein  mutants) possess dull, dark red-brown eyes as opposed to  the bright red eyes of wild-type (wt) flies. hypoxanthine  into  uric  acid,  hydroxypterin to isoxanthopterin, reactions  involving  accumulate  pteridines.  the substrates  XDH  also  1956;  A s a result, rosy hypoxanthine  and  "  mutants  2-amino-4-  (Hadron and Schwink,  The absence of this gene does not greatly  impair fly survival under normal conditions. temperature i s increased  2-amino-4-  Mitchell et al., 1958) and  pteridines, biopterin, and sepiapteridine Graf et al., 1959).  oxidizes  as well as catalyzing many other  hydroxypterin (Hadron and Schwink, 1956; the  In addition to converting  However, i f the growth  to 29 °C, significant increase  i n pupal  11  mortality is observed (Glassman, 1965). The  rosy  locus has been studied extensively.  A host of  spontaneous, chemical and radiation induced mutants have been isolated, and intensive fine region has been performed gene was sequenced al.,  at this  structure analysis  (Cote et al., 1986).  site  of the  In 1987, the Xdh  and the intron/exon boundaries defined (Lee et  1987; Keith et al., 1987).  The Xdh  gene contains 4 exons and  encodes a 1335 amino acid protein of 147 kD. The r 3.4 kb deletion in the coding region of the gene.  5 0 6  mutation is a  It encompasses the  last one-third of the second exon, the entire third and fourth exon as well as about a kb of 3' flanking D N A (see figure 12).  X D H is a  soluble protein that functions as a homodimer. 2.  Purine selection The presence or absence of X D H activity i n Drosophila  determined by growth on food supplemented et al,  can be  with purine (Finnerty  1970). The rosy ~ individuals succumb prior to eclosion, while  wt flies develop normally.  The mode of purine toxicity is unclear.  Purine does not appear to be a substrate for X D H , yet it inhibits the conversion  of hypoxanthine  to xanthine.  A s a result, the  hypoxanthine accumulates i n the malpighian tubules and causes the death of the f l y (Glassman, 1965).  The concentration of purine  required for selection is proportional to X D H activity. titration of the purine concentration, rosy  " mutants  Hence, by  with decreased  enzymes activity can also be distinguished from null mutants.  12  E.  The Drosophila  1.  The composition of the eggshell Drosophila  Eggshell  embryos are unlike the simple,  cells typically transformed by electroporation. outer protective  layers, which  membrane bound  They possess many  together allow  the penetration of  sperm and the exchange of respiratory gases, yet still shield the embryo from mechanical injury and desiccation.  Protective layers  present i n plant cells, yeast and gram positive bacteria are removed prior to transformation The eggshell, 1980).  by electroporation.  outer protective coating of Drosophila consists of five distinct layers  embryos, termed the  (figure 3;  Margaritis  et al.,  From the outer most, they are: exochorion (300-500 nm) endochorion (500-700  nm)  innermost chorionic layer (40-50 nm) 'waxy' layer (0.50 nm) vitelline membrane (300 nm) The The  exo and endochorions are composed primarily of protein.  exochorion is a dense protective coat, while the endochorion is  composed of a rigid network of cavities. with  a i r after  ovulation  and  together  These cavities are filled with  the respiratory  appendages are responsible for gas exchange (Margaritis et al, 1980). The  innermost chorionic layer is polycrystalline i n nature, though its  function is yet undefined. removed with  These three outermost layers are easily  a short hypochlorite  treatment ( H i l l ,  what is termed a dechorionated embryo.  1945), leaving  Figure 3.  A  The  Main Layers of the Eggshell  Drosophila  three dimensional representation of a fragment of the Drosophila eggshell main body indicating the relative orientation of various 2-dimensional views. (Margaritis et al. , 1980)  14  Probably the best physical evidence for the existence of the wax layer comes from Margaritis et al. techniques  (transmission,  (1980), who used ultrastructural  scanning  and freeze-fracture  microscopy) to examine the eggshell of Drosophila. studies, they revealed  electron  Through their  the presence of a thin layer of hydrophobic  plates which were devoid of proteins or other large molecules, and which  produced  smooth  fracture  faces  during  freeze-fracture  studies. The  vitelline membrane is the innermost barrier of the embryo.  It is highly  protienaeous ( 7 4 % ) ,  consisting primarily of Alanine  (29%), Proline (18%), and Serine (17%). via  The proteins are cross-linked  bonds between the meta carbons of tyrosine.  Therefore, the  vitelline membrane cannot be dissolved easily (Petri et al., 1976; Fargnoli and Warning, 1982). 2.  Embryo  permeability  Drosophila  impermeable vapour.  embryos to everything  Even  are essentially a  except  respiratory  closed  gases  system,  and water  dechorionated, the embryos remain impervious to  water soluble molecules.  The primary reason for this is believed to  be a the thin wax layer which is laid down between the chorion and the vitelline membrane just prior to ovulation (Davies, 1947). layer has been compared  to an insects cuticle because of their  functional similarity in waterproofing,  but the two layers originate  from different cell types (Cummings et al., 1971).  The existence of  this layer prohibited embryo fixation as well as in vivo metabolism  and  This  protein  synthesis  which  studies of  required  the  15  uptake of water soluble molecules. this barrier were investigated.  Therefore, methods to remove  Three methods that were found to  remove the wax layer (thus making the embryo permeable to water soluble molecules) were detergent solubilization, solvent extraction and heat removal. Detergents such as Triton-X 100 (TX-100) and sodium dodecyl sulphate (SDS) have been shown to solubilize the wax layer, thus et  making the embryo permeable  to water soluble molecules (Eudy  al.,  The detergents are able to displace the  1969; Sayles et al., 1973).  wax molecules and form micelles, thus removing the wax from the surface of the embryo. Solvents such as heptane and octane have also been used to increase the permeability of Drosophila Zalokar, 1973).  embryos (Limbourg and  Since the hydrophobic wax molecules are more  soluble i n the organic solvent than in an aqueous environment, the solvent  extracts  the wax from  the embryo  surface, leaving an  embryo which is freely permeable to small water soluble molecules. Evidence for the presence of a wax layer was first derived from observations that heating  the Drosophila  embryo  makes i t very  sensitive to the osmotic state of the incubation buffer (King and Koch, 1963).  For example, an embryo placed in a saturated saline  solution developed normally, unless the solution was heated to 45 °C. The increased temperature caused the embryo to lose water rapidly in the hypertonic solution, shrivels up and cease development.  This  observation implies that the wax barrier melted, thus resulting in an embryo that is extremely permeable to water.  16  F.  Scope  of  this  Thesis  The original goal of my thesis was to develop electroporation as Drosophila.  an alternative method for the germline transformation of However,  the protective  layers  of the dechorionated  embryo  (especially, the wax layer present between the chorion and the vitelline  membrane) proved  a greater obstacle to the efficient  formation of electrically induced pores than anticipated. methods to remove investigated.  The  solubilization, results of both experiments  this layer without harming approaches  solvent extraction  examined  are presented here.  the embryo  were  included, detergent  and temperature  the electroporation  Therefore,  effects.  studies and the wax  The removal  17 II.  M A T E R I A L S and M E T H O D S :  A.  Materials  All  reagents  and chemicals  were  purchased  from  MCB  (Matheson Coleman and Bell) Reagents, Aldrich Chemical Company, BDH  Inc., Fisher Scientific Company, or Nichols Chemical Company  Ltd.  The antibiotics, lysozyme, spermine, spermidine, B S A , and  purine were purchased from Sigma Chemical Company.  The Bacto-  tryptone, Bacto-agar, and yeast extract were obtained from The  Difco.  soy flour for the f l y food was made by Stone-Buhr, the agar  from U S B C , the glucose and sucrose from  BDH  respectively, and the methyl-p-hydroxybenzoate was made  by 3 M  and B C  from  and the  Sugar  BDH.  The  Miracloth  by  double-sided  tape  Calbiochem.  The electroporators used were either a Bio-Rad gene  pulser equipped with a capacitance extender or a B R L Cell-Porator. The E. coli  D N A Pol I, Bam H I and the electrophoresis grade agarose  were acquired from Pharmacia. from Dr. D.A. Sinclair. Amersham.  The herring sperm D N A was a gift  The nylon membranes (Hybond N) were by  Nile Blue, Methyl Red and Blue Dextran were purchased  from Allied Chemical, M C B , and Pharmacia respectively.  The pentane  and heptane were from BDH, while the decane was from Sigma. The detergent TX-100 was purchased from J.T. Baker Chemicals Co.  B.  Buffers  The  and  Solutions  composition of the buffers and solutions used  were as  follows: Basic Incubation Media (BIM):  9 m M M g C l , 10 m M M g S 0 , 3 m M 2  4  NaH2P04, 68 m M glutamic acid, 67 m M glycine, 4.1 m M malic  18 acid, 0.1 m M (Limbourg  Sodium acetate, 10 m M  glucose, 5.5 m M  CaCl . 2  and Zalokar, 1973)  50 X Denhardt's:  10 g/L Ficoll, 10 g/L polyvinylpyrolidone, 10 g/L  BSA. Drosophila  mM Drosophila  lysis buffer:  100 m M Tris-HCl (pH 8.0), 50 m M NaCl, 50  EDTA, 0.15 m M  spermine, and 0.5 m M  Ringer's (DR):  spermidine.  110 m M NaCl, 1.9 m M KC1, 2.4 m M  N a H C 0 , 0.8 m M C a C l 0.07 m M N a H P 0 . 3  2)  6 X Gel Loading Buffer:  2  4  0.25% bromophenol blue, 0.25% xylene  cyanol, and 1 5 % Ficoll 400. Hepes Buffered Saline (HBSal):  21 m M HEPES (pH 7.05), 137 m M  NaCl, 5 m M KC1, 0.7 m M N a H P 0 , 6 m M 2  10 X Nick Translation Buffer:  4  glucose.  0.5 M Tris-HCl (pH 7.2), 0.1 M M g C l , 2  1.0 m M DTT, 500 u.g/ml BSA. Nick Translation Elution Buffer: NaCl, 0.25 m M  10 m M Tris-HCl (pH 7.5), 200 m M  EDTA.  Phosphate Buffered Saline (PBSal):  2.6 m M KC1, 1.5 m M K H P 0 , 8 2  4  m M N a H P 0 , 137 m M NaCl. 2  4  Phosphate Buffered Sucrose (PBSuc): potassium phosphate [1.47 m M  0.272 M sucrose, 7 m M K H P 0 , 5.53 m M K H P 0 , (pH 2  4  2  4  7.4)], 1 m M M g C l . 2  PBSuc TN:  PBSuc, 0.05% TX-100, and 0.01% Nile Blue.  PBSuc TX:  PBSuc and 0.05% TX-100.  20 X SSC:  3 M NaCl, 0.3 M sodium citrate pH 7.0.  TBE:  89 m M Tris-HCl, 89 m M boric acid, 2 m M E D T A (pH 8.0)  TE8:  10 m M Tris-HCl, 1 m M EDTA, (pH to 8.0)  1  C.  DNA  Amplification  1.  Bacterial strains The E.coli  and  9 Purification  strain D H 5 a was obtained from Dr. D.A.  Sinclair and  kept as frozen stocks (1 ml aliquots in 1 5 % glycerol at -70 °C). bacteria were grown on SOB 10 mM  NaCl, 2.5 mM  KC1, 10 mM  supplemented with 20 mM g/L yeast extract, 4%  ( 2 % Bacto-tryptone, 0.5% M g C l , 10 mM  glucose) or TB  glycerol, 17 mM  4  achieved by  KH2PO4, and 72 mM  addition of Ampicillin  (SOB  (12 g/L Bacto-tryptone, 24  broth or plates (15 g of Bacto-agar/litre of broth). was  yeast extract,  M g S 0 ) , SOC  2  The  K2HPO4)  Plasmid selection  [25 pg/ml (broth) or  100  and  Spradling, 1983)  and  were obtained from Dr.  CH.  pg/ml (plates)] 2.  Vectors The  vectors pCarnegie  p7t25.1 (Spradling and Newton. pUC8.  Rubin,  20  (Rubin  1982)  pCarnegie 20 contains a full length P-element inserted into The  P-element possesses a polylinker into which the 7.2  Hind III fragment of the rosy  kb  gene was cloned.  p;t25.1 is a vector  containing a 2.9 kb P-element and approximately  1.8 kb of flanking  Drosophila  3.  DNA  cloned into the Bam  HI site of pBR322.  Bacterial transformation The  plasmids were transformed into E.  coli.  D H 5 a competent  cells essentially as outlined by D. Hanahan (1985), with the following modifications.  A  200  p i aliquot of frozen competent cells  thawed, 2-4 ng of plasmid DNA for 30 min.  was  was added and the solution set on ice  The cells were given a heat shock (90 seconds at 42  and immediately placed back on ice. SOC suspension incubated at 37 °C for 45 min.  °C)  (800 pi) was added and the The bacteria were plated  20 on SOB-Amp plates and grown overnight at 37 °C.  A liquid culture  was then prepared by inoculating 5 ml of TB-Amp  with  a single  isolated colony from the plate and grown at 37 °C for 12-16 hrs. 1 ml stocks were prepared in 1 5 % glycerol and stored at -70 °C. 4.  Plasmid a.  isolation  Mini  preps  Plasmid D N A was isolated from overnight cultures (5 ml of TBAmp inoculated with a single isolated colony and grown at 37 °C for 12 hrs) by the alkaline lysis method (Birnboim and Doly, 1979) as follows: mM  100 p i of ice cold Glucose-Tris buffer [50 m M  E D T A , 25 m M  Tris-HCl (pH 8.0), and 4 mg/ml lysozyme] was  added to the pellet of 1.5 ml of culture. room  glucose, 10  After a 5 min. incubation at  temperature, 200 p i of fresh, ice cold N a O H solution (0.2 N  NaOH, and 1 % SDS) was added, the tube inverted to mix, then placed on ice for 5 min. Subsequently, 150 p i of ice cold K O A c solution (3 M potassium, and 5 M  acetate) was added, the tube vortexed gently i n  an inverted position and incubated on ice for 5 min. The solution was spun for 5 min. in a microfuge. The supernatant was transferred to a fresh tube and extracted with  one-half volume of Tris-equilibrated phenol [phenol  repeatedly with 0.1 M  extracted  Tris-HCl (pH 8.0) until the p H of the phenol  was above 7.6] and one-half volume of chloroform.  The aqueous  phase was transferred to a fresh tube and the D N A precipitated with 2 volumes of cold 9 5 % ethanol for 10 min.  The D N A was pelleted,  washed twice ( 9 5 % then 7 0 % ethanol), dried and finally resuspended in 20-50 p i of TE8 [10 m M Tris-HCl, 1 m M EDTA, (pH 8.0)] containing 20 pg/ml RNase A.  21 b.  Large scale plasmid isolation  A 500 ml culture was prepared by inoculating TB-Amp 0.5 ml of a 5 ml culture, and incubating at 37 °C overnight.  with  The cells  were pelleted in 250 ml tubes (6 K for 5 min. in a G S A rotor), the supernatant poured off, and 5.0 ml of cold Glucose-Tris solutions are the same as for the mini preps) added. was  transferred  to a fresh  centrifuge  buffer (all  The suspension  tube to which  20 mg of  lysozyme was added and the tube incubated at room temperature for 5 min.  10 ml of cold N a O H solution was then added, the solution  mixed by inversion and placed on ice for 10 min. KOAc  solution was then mixed i n by vortexing  7.5 ml of cold gently, and the  solution was centrifuged (10 K for 20 min. at 4 °C, in a SS34 rotor). The  supernatant was transferred to a fresh tube and extracted  with 8.0 m l of Tris-equilibrated phenol and 2.0 ml of chloroform for 10  min. on a rotating wheel.  centrifugation (10 K for 10 min. aqueous phase was transferred  The extraction was followed at 15 °C, i n a SS34 rotor). to a fresh tube, the nucleic  by The acids  precipitated (0.6 volume of propanol-2 at room temperature for 10 min.), and pelleted by centrifugation (8 K for 10 minutes at 20 °C, in a SS34 rotor).  The pellet was washed with 9 5 % ethanol, dried, then  resuspended i n 400 p i TE8 supplemented with 200 p i of 250 m M EDTA. C s C l ( 4 . 2 g) and 300 p i of EtBr (10 mg/ml) were added, the solution vortexed and incubated on ice (in darkness) for 15 min.  The  R N A was pelleted by centrifugation (10 K for 10 min. at 4 °C, in a SS34 rotor).  A n additional 200 p i of EtBr was added to the  supernatant, and the solution transferred to a Beckman  quick-seal  22 centrifuge tube. After centrifugation (50-55 K for 12-17 hrs at 20 °C, in a VTi65 rotor), the lower of the 2 resulting bands was withdraw with a 1 ml syringe equipped with a 21 G 1/2 needle. was  The solution  placed i n a tube to which 2 volumes of d H 2 0 were  Extraction  of the EtBr  was then performed  with  added.  water-saturated  butanol (8-9 extractions), the final aqueous phase being transferred to a 30 m l Corex tube and the D N A precipitated with 0.1 volume of 2.5 M  Sodium acetate (pH 5.2), and 2 volumes of cold 9 5 % ethanol  overnight at -20 °C. The tube was centrifuged (5 K for 20 min. at 4 °C, i n a SS34 rotor), the pellet washed once with 9 5 % ethanol and twice with 7 0 % ethanol, dried, then resuspended in 200-400 u,l TE8 and  transferred  to a  microcentrifuge  reprecipitated with 0.1 volume of 2.5 M  tube.  The  DNA  was  Sodium acetate (pH 5.2) and  2.5 volumes of cold 9 5 % ethanol, pelleted by centrifugation (10 min. in a microfuge).  The pellet was washed once with 9 5 % ethanol and  once with 7 0 % ethanol, dried, and finally resuspended in 50-100 u.1 TE8.  Purity of the D N A was determined by A 260/280 nm and mini  gel electrophoresis.  D.  Southern  Analysis of Drosophila  1.  Rapid phenol extraction of genomic D N A Genomic D N A  (1986),  with  Genomic D N A  was extracted from flies as outlined in Jowett  the following modifications: 200-400  flies  (equal  numbers of males and females) were ground to a powder in a small mortar containing  approximately  2 m l of liquid  nitrogen.  The  resulting powder was scraped into a 30 ml Corex tube containing 1  23 ml/100 flies of lysis buffer [100 m M Tris-HCl (pH 8.0), 50 m M 50 m M  E D T A , 0.15 m M  spermine, and 0.5 m M  NaCl,  spermidine], and 10  u.1/100 flies of Proteinase K (10 mg/ml in 5 0 % glycerol) was added. The solution was then incubated at 37 °C for 2 hrs (with occasional mixing), extracted once with  1 volume of Tris-equilibrated  phenol,  then twice with one-half volume Tris-equilibrated phenol and onehalf volume  chloroform/isoamyl  alcohol ( I A A ) (10:1), and finally  with one volume of the chloroform mixture. The  resulting  aqueous  phase  containing 0.1 volumes of 2.5 M  was transferred to a  tube  Sodium acetate (pH 5.2) and 2  volumes of cold 9 5 % ethanol. The D N A was precipitated at -20 °C for 1-2 hrs. The D N A was pelleted (8 K for 10 min. at 4 °C, in a SS34 rotor),  the pellet  briefly  dried  i n a vacuum  desiccator and  redissolved in 400 u.1 TE8. After transfer to a microcentrifuge tube, 100 u.g/ml of RNase A was added and the tube incubated at 37 °C for 30 min.  Extractions with phenol/chloroform  were performed. with  ethanol  and chloroform  alone  The D N A was precipitated, pelleted, washed  twice  ( 9 5 % then  7 0 % ) , dried  as before,  and f i n a l l y  resuspended i n 50-100 u.1 TE8. The concentration of the D N A was estimated by assay on a mini agarose gel using bacteriophage X D N A as a standard. 2.  Enzymatic digestion of genomic D N A Restriction  enzyme  digests  of Drosophila  melanogaster  genomic D N A were usually carried out i n a total volume of 200 u.1. Approximately restriction  2-5 j i g of D N A were digested with 2-3 units/ng of  enzyme using the buffer supplied by the manufacturer  24 supplemented with 0.5 m M  spermine) at 37 °C for 2-5 hrs (complete  digestion was monitored by loading 10 p i and 2 p i loading buffer on a 0.7% mini agarose gel).  The restricted D N A  was then ethanol  precipitated, washed, dried and resuspended in 15 p i TE8. Loading buffer (5 pi) was added, and the D N A loaded on a 0.7% agarose gel (1 X TBE, 0.7 mg/ml EtBr).  The gel (25 x 20 cm) was run for 10 min. at  100 V, then at 30-50 V for 18-24 hrs. 3.  Southern  Analysis  a.  Southern  The  DNA  transfer  i n the agarose  gel was transferred  membrane by the method of Southern (1975).  to a nylon  The gel was covered  with denaturing solution (1.5 M NaCl, 0.5 M NaOH) and incubated at room temperature for 1 hr (with gentle shaking).  The gel was rinsed  with dH20, then submerged in neutralizing solution (1 M (pH  8.0), and 1.5 M  Tris-HCl  NaCl) for 1 hr(room temperature and gentle  shaking), and rinsed again.  The gel was placed on a piece of filter  paper supported by a glass plate or a sponge in a dish of 10 X SSC . A  nylon (Hybond-N) filter cut to size, 2-3 pieces of filter paper  soaked i n 10 X SSC, a 2-3 inch stack of paper towels, a glass plate and finally lead weights were place on top of the gel. The transfer was allowed to proceed at room temperature for 8-12 hrs.  The filter  was removed, rinsed in 6 X SSC, wrapped in Saran Wrap®, exposed to U V light for 3 min. and stored at -20 °C till required. b.  Preparation of nick-translated probe  A 50 p i solution containing the following: 5 p i 10 X Nick Translation buffer, 2.5 mg/ml BSA, 1.0 p i 0.5 M (3-mercaptoethanol,  25 2.0 p i 0.5 m M dGTP, dTTP, and dCTP, 2.0 u l 35 u M dATP, 3-5 p i [ a 32p]dATP, 0-1 p i DNase I (10 pg/u.1), 1.0 p i 10 m M C a C l , 1 u.g plasmid 2  DNA, and 10 units E. coli. Pol I was incubated at 15-16 °C for 1.5 hrs. The reaction was stopped by the addition of 150 p i of 1 % SDS/10 m M ETDA. The solution was heated at 65 °C for 10 min., then 5 p i E. t R N A (10 ng/ml) were added.  coli  The probe was loaded onto an L K B  Ultragel A c A 54 column, and eluted with Nick Translation Elution Buffer [10 m M Tris-HCl (pH 7.5), 200 m M NaCl, 0.25 m M EDTA]. Sixdrop fractions were collected while monitoring the separation Geiger  counter.  The tubes  containing  the most  with a  incorporated  radioactive were pooled, their combined volume measured and the radioactivity 10  7  assessed by the Cerenkov method.  Incorporation of  cpm/pg D N A was typical. c.  Hybridization of filters  The genomic D N A laden filter was placed in a heat sealable bag with  10 m l of Prehybridization solution (6 X SSC, 0.5% SDS, 5 X  Denhardt's,  and 100 pg/ml  prewarmed to 68 °C.  denatured  herring  sperm  DNA)  The bag was sealed and the filter incubated at  68 °C for 4 hrs. The nick-translated probe was then added to the bag, the bag resealed and incubated at 68 °C for a further 12-18 hrs. d.  Washing of filters  The filter was removed from the bag, placed in a solution of 1 X SSC and 0.5% SDS prewarmed to 68 °C and incubated at 68 °C for 45-60 min.  The solution was changed and a further 1 hr incubation  was performed. Two 1-hour washes in 0.1 X SSC and 0.5% SDS at 68 °C were then done, and the filter was wrapped in Saran W r a p ® and  26 exposed  to X-ray  film  for 1-3 days depending  on radioactivity  retained on the filter.  E.  Drosophila  1.  Drosophila  Embryos melanogaster  strains  Isogenic wild-type Oregon R flies were obtained from Dr. G.M. Tener. The r 0 6 / 5 0 6 mutants were obtained from V.K. L l o y d (UBC). 5  r  Both strains were maintained i n sponge stoppered  160 ml Corning®  glass dilution bottles containing 30 ml of enriched fly food (1 litre of tap water, 100 g soy flour, 20 g yeast extract, 17 g agar, 1 g citric acid, 9 g trisodium citrate, 40 g glucose, 40 g sucrose, 17 ml 1 0 % methyl-p-hydroxybenzoate i n 9 5 % ethanol, and two of the following antibiotics: 20 mg  streptomycin,  10 mg  tetracycline, or 30 mg  ampicillin). 2.  Embryo collection and dechorionation Flies 5-8 days old were placed i n small milk bottles.  The  opening was covered by a small Petri dish containing 1 % agar lightly spread with yeast paste (yeast powder with 2 % acetic acid and 5 % ethanol) placed inside an adapter. The collection plates were changed every  25-35 minutes (after an initial 2-3 hour prelay where they  were changed about every hour).  The embryos were loosened  from  the agar with d H 2 0 and a paint brush, and poured into a 1.5 cm diameter  buchner funnel lined  embryos  were  dechorionated  with  a disk of Miracloth.  by washing  them  with  3%  hypochlorite (Javex or Sunbrite), then were rinsed with dt^O.  The  sodium  27 3.  Assessment of embryo survival The ratio of  within  48  recovered  the number of first instar larvae which hatched  hrs post  treatment to the total  after treatment  Survival  to  subsequent  was life  used  number  to assess  cycle  stages  of embryos  embryo  was  survival.  evaluated  by  transferring of the larvae to vials of enriched food and the number of  adults  which  pupated  and  eclosed  counted.  Fertility  was  determined by mating individual virgins to 5 virgins of the opposite sex.  Matings which produced progeny were deemed  to be from  fertile individuals. 4.  Assessment of embryo  permeability  Embryo permeability was assessed by their ability to take up a water soluble dye such as Nile Blue or Methyl Red. became visibly coloured, were deemed of permeable embryos was coloured  permeable.  Embryos that The percentage  calculated by dividing the number of  embryos by the total number  of embryos recovered  after  treatment. 5.  Tape-mounted Two  were  embryos  layers of double-sided  placed  dechorionated  on  a glass  slide.  tape cut into thin (1 mm) The  Miracloth  strips  containing the  embryos was placed on the slide and the slide placed  in the bottom half of a petri dish lined with a disk of filter paper moistened with dH^O or PBSuc. (This dish when covered with a lid is referred to as a humid chamber.)  The embryos were then mounted  side by side on the tape in groups of 25-50, using a paintbrush. lid was then placed on the. dish.  The  Individual tapes were treated as  28 desired, placed on a new slide, covered  with halocarbon  o i l and the  slide then placed back in a humid chamber. 6.  Non-mounted  embryos  Dechorionated  embryos were washed  the appropriate After  off the Miracloth with  buffer (containing 0.05% TX-100) into a cuvette.  the desired treatment,  the solution containing the embryos  was poured out of the cuvette into the buchner funnel lined with a fresh disk of Miracloth, the cuvette rinsed out thrice with buffer and the embryos rinsed briefly with buffer. on a slide, covered  with halocarbon  chamber, or put directly  The cloth was then placed o i l and placed  onto the moistened  i n a humid  filter paper i n the  chamber. a.  Detergent-treated  Dechorionated  embryos  embryos were washed  off the Miracloth with  PBSuc (containing the desired amount of TX-100), into a 0.2 cuvette.  cm  The embryos were incubated for 20 minutes, then collected  on Miracloth, rinsed with buffer and placed (PBSuc).  The  embryos  were  allowed  in a humid chamber to develop  at  room  temperature. b.  Solvent-treated  Dechorionated cm  embryos  embryos were washed off the Miracloth into a 1  diameter cup made of 100-mesh stainless steel and the cup  submerged i n about 2 ml of solvent for the desired time. solvent was  drawn out of the cup with  embryos rinsed with buffer.  paper  The  towels, and the  The embryos were then rinsed out of  the cup and into the lined funnel with buffer, and the embryos  29 treated as above (non-mounted embryos), c.  Heat-treated  Dechorionated  embryos  embryos were washed  into a 0.2 cm  cuvette  with 500 p i of PBSuc TN. The cuvette was placed in a water bath at a specified temperature,  and the solution incubated for 20 minutes.  The embryos were then collected on Miracloth, rinsed with PBSuc, and placed in a humid chamber (PBSuc). to develop 7.  at room  The embryos were allowed  temperature.  Electroporation of embryos Dechorionated  embryos were either rinsed off the Miracloth  into a 0.2 or 0.4 cm cuvette with the appropriate buffer, or tapemounted and the tape placed down the side of the cuvette. embryos were preincubated  at room  The  temperature for 1-5 minutes,  pulsed at the desired field strength and capacitance, then  incubated  a further 1-15 minutes at room temperature (any embryos not in solution were removed).  The suspended embryos were collected on  Miracloth, and rinsed with buffer. removed  from  The tape-mounted embryos were  the cuvette, the tape  embryos covered  with halocarbon  oil.  placed  on a slide, and the  Both sets of embryos were  then placed i n a humid chamber (PBSuc) and allowed to develop at room  temperature.  F.  Purine  1.  Purine  Selection  titre  Vials containing five male and ten female flies of the following strains were  prepared:  30 1.  The  +/+ X +/+ 506/ 506 x  r  506/ 506 X  +/+  2.  r  3-  r  r  r  5 0 6  /r  5 0 6  first two crosses produce homozygous  wt and r  respectively, while the third cross produces F i +/r The vials each contained 5 ml of enriched food.  506  F i flies  5 0 6  heterozygotes.  The flies were left to  lay embryos for 2-3 days then transferred to a fresh vial.  100 p.1 of  purine solution (the desired quantity of purine (w/v) dissolved in d H 2 0 ) was added to the vacated vial. every 2-3 days for 8 days.  The flies were transferred  The vials were left at room  temperature,  and the number of adults which eclosed from each vial was tallied. 2.  Purine selection of putative transformants Ten males and 20 females  from each putatively  transformed  line were mated in glass vials containing 5 ml of enriched food.  The  flies were allowed to lay embryos for 2 days then were transferred to a fresh vial. vacated vial.  100 u.1 of purine (0.2% (w/v)) was added to the The flies were transferred every 2 days for 8 days.  The embryos were allowed to develop  at room temperature.  Lines  surviving the purine selection process were scored as containing a wild-type Xdh  gene.  31 III.  R E S U L T S and DISCUSSION  A.  Effect of the Exposure of Drosophila Electrical Current  1.  Survival of embryos exposed to increasing field strengths Dechorionated  field strengths.  Embryos to an  embryos were exposed to a range of electrical Increases i n the field strength lead to corresponding  decreases i n the ability of the embryos to survive subsequent to the pulse  (figure 4).  range investigated. embryos hatched. not  The survival decreased fairly  steadily over the  A t 10 kV/cm only about 1 0 % of the treated The survival of embryos mounted on tape (data  shown) was often considerably  lower  (almost  half) than for  embryos suspended i n 0.05% TX-100. 2.  Permeability of embryos exposed to increasing field strengths Dechorionated  dye  Methyl  embryos became receptive to the uptake of the  Red, only at high field strength (figure 5).  To achieve  dye uptake above background, over 10 kV/cm at a capacitance of 25 (iFd was required.  Above this value, greater than 7 5 % of the treated  embryos became permeable to the water soluble dye. than 1 0 % survived the treatment.  However, less  In contrast, embryos pulsed over  a range of voltages in the presence of Blue Dextran or [ H ] - D e x t r a n , 3  were unable to take up detectable amounts of dye or radioactivity (data not shown). 3.  Survival of embryos exposed to increasing capacitance Dechorionated  embryos were exposed to a field  kV/cm over a range of capacitances Porator.  strength of 1  (0-1980 u.Fd) in a B R L Cell-  As expected, survival decreased steadily as capacitance and  therefore x increased (figure 6).  When capacitance was varied over  Figure  4.  The Effect of Increasing Embryo Survival.  Field  Strength  on  5 0 6 / 5 0 6 flies (five to six days old) were placed i n a laying bottle i n the dark, and allowed to lay eggs for 2 hrs. Subsequently, the egg laying plates were changed every 30 min.. The embryos from each collection were dechorionated, rinsed with d H 2 0 , then rinsed off the Miracloth into a 0.2 cm cuvette with 500 p i of PBSuc TX. The cuvette was left at room temperature for 5 min., the embryos resuspended by tapping and the suspension r  r  pulsed at the indicated field strength (25 pFd). T's ranged from 1.7 (10 kV/cm) to 2.6 msec. (2.5 kV/cm). The embryos were incubated a further 15 min. at room temperature (any eggs not i n solution were removed). The embryos were collected on Miracloth, rinsed with PBSuc and placed i n a humid chamber (PBSuc). The percentage of embryos that survived to first instar larvae to hatch within 48 hrs of the treatment is given.  Figure  5.  The Effect of Increasing Field Strength on E m b r y o Survival and Permeability to the Dye Methyl Red. fii  (five  to six days old) were placed i n a laying bottle i n the dark, and allowed to lay eggs for 2 hrs. Subsequently, the egg laying plates were changed every 30 min.. The eggs from each collection were dechorionated, rinsed with d H 2 0 , then rinsed off the Miracloth into a 0.2 cm cuvette with 500 pi of PBSuc T X containing Methyl Red (0.01% w/v). The cuvette was left at room temperature for 5 min., the embryos resuspended by tapping and the suspension pulsed at the indicated field strength r  5 0 6 / 5 06 r  e s  (25 uPd). T's ranged from 1.3 (12.5 kV/cm) to 2.1 msec. (5 kV/cm). The embryos were incubated a further 15 min. at room temperature (any eggs not i n solution were removed). The embryos were collected on Miracloth, rinsed with PBSuc and placed in a humid chamber (PBSuc). The number of embryos collected after treatment (coloured and not) was recorded, as was the number of larvae that hatched within 48 hrs of the treatment.  34  100  Capacitance (uFd)  Figure 6.  The Effect of Increasing Capacitance on E m b r y o Survival and Permeability to the Dye Nile Blue.  fii (five to six days old) were placed i n a laying bottle i n the dark, and allowed to lay eggs for 3 hrs. Embryos were then collected at 30 min. intervals. The embryos were dechorionated, rinsed with dH20, washed into a 0.4 cm cuvette using 900 j i l PBSuc TN. The eggs were incubated for 5 min., and pulsed at 400 volts (1 kV/cm) on the low resistance setting ( B R L Cell-Porator) at the indicated capacitance. After a 15 min. postpulse incubation, the eggs were collected on Miracloth, rinsed with PBSuc, placed i n a humid chamber (PBSuc) and allowed to develop at room temperature. The number of embryos collected after treatment (coloured and not) was recorded, as was the number of larvae that hatched within 48 hrs of the treatment. r  506/ 506 r  e s  35 a range of field strengths, the typical survival curve which showed a decreased  survival  corresponding  to an increased  above), was observed for each capacitance corresponding  downward  voltage (see  (figure 7).  However, a  shift of the survival curve  accompanied  each increase in capacitance. 4.  Permeability of embryos exposed to increasing capacitance Dechorionated  embryos were pulsed (1 kV/cm) in PBSuc T N at  various capacitances  (figure 6).  Permeability to the dye became  extensive at 1180 p F d and above.  A t this point, about 8 0 % of the  treated embryos were able to take up the Nile Blue.  Interestingly,  increasing the capacitance from 1180 to 1980 p F d did not show any further significant increase i n permeability. 5.  Survival of embryos exposed to an electrical current in the presence of D N A Pulsing of embryos i n the presence of D N A did not reveal any  significant change i n survivability of the treated individuals (figure 8).  A  slight decrease i n survival was observed when either the  voltage or the capacitance  (data not shown) was varied.  However,  this is more likely a variation among trials (see below), than due to the presence of the DNA. 6.  Discussion An  electrical pulse has been used to introduce water soluble  macromolecules into a number 1988;  of cell types  Shigekawa and Dower, 1988).  Drosophila  melanogaster  (reviewed  by Potter,  The above results indicate that  embryos also become permeable to water  soluble dyes after electroporation.  A t high field strength (10 kV/cm  36  100  0  2000 Field  Figure 7.  4000 Strength  6000  (V/cm)  The Effect of Varying Capacitance on the Field Strength Survival C u r v e .  fii (five to six days old) were placed i n a laying bottle i n the dark, and allowed to lay eggs for 3 hrs. Embryos were then collected at 30 min. intervals, dechorionated, rinsed with dF^O, and mounted on tape i n groups of 25 (5-6 tapes/collection). Embryos older than approximately 1 hr were not used. The slide containing the mounted embryos was placed i n a developing chamber (PBSuc) until the tape was required (0-5 min.). The tape was placed down the side of a 0.4 cm cuvette containing 800 u.1 PBSuc, and preincubated for 1 min.. The embryos were then pulsed at the indicated field strength, and incubated one more r  506/ 506 r  e s  minute. T's were typically 0.2 (l.OpFd), 0.5 (3.0 u.Fd) and ranged from 3.1 to 4.6 msec. (25.0 u.Fd). The tape was then removed from the cuvette, placed on a new slide and covered with halocarbon o i l . The slide was placed back i n a humid chamber (dH20) and the embryos allowed to develop. The % survival was calculated from the number of embryos mounted i n relation to the number of larvae that hatched within 48 hrs.  37  100  oH  i  1  0  .  2000 Field  Figure 8.  1  4000 Strength  •  1  6000  i  8000  (V/cm)  The Effect of the Presence of D N A During Pulsing on Survival of Embryos Exposed to an Electrical Current.  to six days old) were placed i n a laying bottle i n the dark, and allowed to lay eggs for 3 hrs. Embryos were then collected at 30 min. intervals. The embryos were dechorionated, rinsed with d H 2 0 and mounted on tape (20 eggs/tape, 3-4 tapes/collection). The tapes were placed individually down the side of a 0.2 cm cuvette containing 400 p i of PBSuc (no D N A ) or Electroporation Buffer (10.3 pg/ml pCarnegie 20, 0.545 pg/ml p7t25.1 i n PBSuc), incubated for 30 seconds i n the r  506/ 506 r  fii  e s  (five  buffer and pulsed at the desired field strength (25 pFd). T's were usually approximately 1.9 msec. Forty-five seconds post pulse, the tape was removed from the cuvette, placed on a glass slide, and the embryos covered with halocarbon oil. The slide was then placed back i n a humid chamber (dH^O). Embryos older than 1 hr were discarded. The percentages survival was calculated from the number of embryos mounted (after aging) i n relation to the number of larvae that hatched within 48 hrs of the treatment.  38 or greater) or high capacitances  (800 u.Fd or greater), significant  numbers of the treated embryos become permeable to dyes such as Methyl Red or Nile Blue. than  20%  of  the  However, under these conditions  embryos  survived  the  treatment.  fewer These  observations brings up two questions: 1. Why  is such a high voltage required to make the embryos  permeable to the water soluble dye? 2. The  What is causing embryo death at low field strength? criterion  for the extreme field  uptake is interesting. (Neumann et al.,  strength to produce dye  According to the equation A V m a x = 1.5 E r 0  1982), the critical membrane breakdown potential is  proportional to the radius of the cell (r).  Smaller cells therefore  require larger field strengths (E) for membranes breakdown to occur. Electroporation of the large mammalian  cells typically occurs at < 1  kV/cm (Andreason and Evans, 1988), while for the small bacteria protoplasts, about 6 kV/cm  is required (Skigekawa et al.,  However, the size of the Drosophila than most cells commonly required  an extremely  observed.  high  embryo is significantly larger  transformed field  1988).  by electroporation, yet they  strength for dye uptake to be  This inconsistency presumably results from the remaining  layers of the Drosophila higher field  eggshell, in particular the wax layer.  A  strength would presumably be required to disorganize  the compact wax molecules, than the more fluid phospholipids in a typical bilayer structure. Therefore electroporation  the extraction of this should  protective layer prior  aid in the electroporation process.  to Its  39 removal would make the embryo permeable to small water soluble molecules, and required  as  a result, should  to create  molecules (such Transformation then be  the  do  not  critical  voltage  molecular  weight  to move through the vitelline membrane. de-waxed  re-examined.  membranes  the  pores large enough for high  as DNA) of  decrease  embryo by  electroporation  This, of course, assumes that the pose  another  barrier  to  the  could  vitelline  electroporation  process. The It was  death of the embryos pulsed at lower voltages was  puzzling.  expected that the electric field would have little effect on cell  survival until the critical breakdown potential of the membrane exceeded.  However, in the above experiments, survival consistently  declined with increasing voltage (even at very low parameters). the  was  embryos  suggesting  were  not  that pores had  permeable not formed.  to  water  One  soluble  Yet,  molecules  possible explanation for  this phenomenon is that regardless of the integrity of the membrane, the electric field was The  affecting the development of the embryo.  movement of charged molecules in an electric field is well  documented and within an  characterized.  Hence, the idea of it taking  intact cell is not revolutionary.  nature of D N A  place  Similarly, the  charged  is important in the electroporation process.  It has  been shown that although the pores formed by  the electrical current  are important to this technique, the electrophoretic movement of the charged D N A et al, 1988). plasmid DNA  through the pores, is equally important (Winterbourne Electrical currents  have also been used to extract  from cells (Calvin and Hanawalt, 1988).  40 With this i n mind, it is possible that a high electric field could result  i n electrophoresis  of developmentally  important  molecules,  such as morphogens (Manseau and Schupbach, 1989), or even the nuclei themselves.  Such movement could result i n these molecules  being absent from their site of action at the required time.  This type  of mass electrophoresis would therefore have dire consequences on embryonic  development.  B.  Electroporation  1.  Survival of electroporated embryos from embryo to adult The  of  Drosophila  Embryos  effect of the presence of D N A in the electroporation buffer  on the development of embryos exposed to an electrical current was examined.  Exposure of embryos to an electrical pulse in the absence  of D N A did not greatly effect their ability to develop to adults i f they survived to the first instar larval stage (figure 9).  On average, in the  absence of D N A , 8 1 % of the individuals able to hatch after being exposed to a field strength from 0 to 7.5 kV/cm, subsequently went on to eclose.  When D N A was present in the electroporation buffer  during pulsing, the ability to develop from larvae to adult appearred to depend on the magnitude of the pulse.  A t low field strength, 6  kV/cm and below, survival to eclosion (75%) was similar to embryos pulsed i n the absence of D N A (81%).  However, above 6 kV/cm, a  significant number of the larvae did not develop into adults when DNA  was present i n the electroporation buffer.  eclosed from the surviving larvae was only 4 3 % .  The average  %  41  Figure 9 .  The Effect of the Presence of D N A During Pulsing on the Development of E m b r y o to A d u l t .  to six days old) were placed i n a laying bottle in the dark, and allowed to lay eggs for 3 hrs. Embryos were then collected at 30 min. intervals. The embryos were dechorionated, rinsed with d H 2 0 and mounted on tape (20 eggs/tape, 3-4 tapes/collection). The tapes were placed individually down the side of a 0.2 cm cuvette containing 400 p i of PBSuc (no DNA) or Electroporation Buffer (10.3 pg/ml pCarnegie 20, 0.545 pg/ml p7i25.1 i n PBSuc), incubated for 30 seconds in the r  506/ 506 r  fii  e s  (five  buffer and pulsed at the desired field strength. T's were typically approximately 1.9 msec.Forty-five seconds post pulse, the tape was removed from the cuvette, placed on a glass slide, and the embryos covered with halocarbon oil. The slide was then placed back i n a humid chamber (dH20). Embryos older than 1 hr were removed. Embryos that hatched within 48 hrs of treatment were rescued from the humid chamber and transferred to enriched food. The larvae were allowed to develop at room temperature. The number of flies to eclose in relation to the number of larvae placed in the food vial is given.  42 2.  Phenotypic To  selection of putative transformants  screen  for transformants,  individuals  treatment, were mated individually to r from  these  crosses were scored  5 0 6  /r  5 0 6  that eclosed flies.  The progeny  for eye colour phenotype.  results of the screen are recorded i n Table I.  after  The  From these data, it  appeared that increases in field strength had given rise to increased transformation.  However, the presence  of F i wild-type progeny i n  control lines suggests other possibilities (see discussion). 3.  Chemical a.  analysis of putative transformants  Purine selection titre  The presence of an active Xdh  gene can also be determined by  growth of the larvae on food supplemented with purine. The r  5 0 6  strain and the wt strain were allowed to lay embryos on  normal food for 3 days.  They were then removed and 100 u.1 of  purine was added to the vials containing the embryos. wild-type (+/+) and heterozygous cross between r  5 0 6  /r  5 0 6  Homozygous  ( + / r ) flies (the F i generation of a 506  and wt) were able to survive on the purine  supplemented food at all concentrations examined (figure 10). contrast, although  the homozygous  mutants  (r506/ 506) r  In  produced  good larval growth, few flies eclosed even at the lowest concentration of purine used (0.1%). B y 0.2% purine none eclosed. b.  Purine selection of putative transformants  From the above titre, 0.2% purine was adequate to eliminate all non-transformed phenotypically  progeny. determined  are given i n Table I.  The selection  was performed  putative transformants  on the  and the results  Seven putative transformants, originating from  Table I. The Analysis of the Electroporation of r pCarnegie 20 Field No. % Strength Treated Hatched Eclosed Fertile (V/cm) 0 5000 6000 7000 8000  74 92 106 277 238  64 35 . 18 15 10  86 75 50 46 47  100 75 67 73 62  No. F wt 0  2 0 1 1 2  506  /r  506  Embryos with  No. Fiwt 2 2 3 4 5  No. No. Purine Southern Selected Analysis ND 0 2 1 4  ND 0 0 0 0  506/ 506 fli prelaid in collection bottles for 3 hrs. The eggs were then collected at 25-30 min. intervals. The eggs were dechorionated, rinsed with d H 2 0 , mounted on tape and kept in a developing chamber (dH20) until needed. Individual tapes were placed down the side of a 0.2 cm cuvette containing PBSuc, incubated for 15 seconds and pulsed at the indicated field strength (25 u,Fd). Thirty seconds post pulse, the tape was removed, placed on a glass slide, the embryos covered with halocarbon oil, embryos older than 2 hrs removed, and the slide placed in a developing chamber (dH^O). Embryos that hatched within 48 hrs of treatment were transferred to standard food and allowed to develop at room temperature. Eclosing adults were scored for eye colour and mated individually to r / r virgins. Fi adults were also scored for phenotypic expression of XDH then brother/sister mated, and their progeny subjected to purine selection (see Materials and Methods). Fly lines which gave positive results to the chemical selection were examined by Southern Analysis. ND = no data available. r  r  es  w e r e  506  506  44 1200  #wt/wt  0  0.1 0 . 2 0 . 3 0 . 4 %  Purine  1200 1000 800 600 400 200 -  W §  , , , i  r  . . . # rosy/Wt  pi Ill m  0 0  0.1 0.2 0.3 0.4 %  Purine  1200 1000 -  # rosy/rosy  0  0.1 0 . 2 0 . 3 0 . 4 %  Figure  10.  Purine  Purine  Selection  Titre.  Five male and ten female flies were placed in a glass vial containing 5 ml of enriched food. The flies were left to lay eggs for 2-3 days then transferred to a fresh vial. 100 | i l of dH20 containing the indicated percentage of purine (w/v) was added to the vial containing the developing larvae. The vials were left at room temperature, and the number of adults to eclose were counted. The numbers reported are a summation of duplicate experiments run over 10 days (8 vials in total). +/+ = wild type Oregon R male and female matings, r/r = r / r male and female matings, and r/+ = 5 0 6 / 5 0 6 males mated to wild type Oregon R females. 5 0 6  r  r  5 0 6  45 5 Fo individuals were found: 2M2, and  6F2.  Lines  2M3,  4M1,  6Mla, 6Mlb, 6Mlc,  of these flies were maintained  by brother/sister  matings. 4.  Molecular  analysis of putative  Southern analysis was from the  The  performed  seven 'transformed'  homozygotes. mating  A  on  lines, as  genomic D N A  well as wt  /r  5 0 6  5 0 6  gene at a undefined  used , r  5 0 6  (brother/sister) created  r  /r  5 0 6  5 0 6  / * , and */*.  the 5' end of the Xdh  The  RI bands of a l l putative transformants  band is common for both r  and  5 0 6  5 0 6  locus.  wild-type genes and  gene (see figure 12). kb Eco  5 0 6  figure can  be  9.2  kb  The  comes from  In the wild-type gene,  RI fragment (3' end)  However, in r  Xdh  (Where * represents the  explained as originating from either a wt or r  there is also a 4.6  heterogeneous  locus in the putative transformants)  suggests that the Eco  pCarnegie 20.  and  extracted  representative of the results is given in figure 11.  strategy  populations of r  transformants  that hybridizes to  , the 3.4 kb deletion includes the 3'  Eco RI site. As a result, the next Eco RI site (0.6 kb 3') becomes the 3' end of the second band, creating a 1.8 kb fragment. 5.  Discussion The  presence of D N A  in the electroporation buffer had  little  effect on the development of the flies from larvae to adult except at high field strength. embryo  during  development genes.  due  This suggests that the DNA  the to  high  voltage  pulse,  integration into  may and  interfered  developmentally  This however seems unlikely since no  isolated, and  have entered the with  important  transformants  were  as shown above, the embryos were not permeable to  46  Figure  11.  Southern Analysis of Transformants.  Putative  Genomic D N A from lines created by brother/sister . mating of suspected transformants were digested with E c o RI and run out on a 0.7% agarose gel (25 x 20 cm). The gel was cut in two length wise, and the D N A was transferred to nylon membranes (Hybond-N). The membranes were hybridized individually with pCarnegie 20 (nick-translated with [ a P ] d A T P ) at 68 °C for 16 hrs. The membranes were then washed at 68 °C with 1 X SSC, 0.5% SDS, then 0.1 X SSC, 0.5% SDS. The membranes were exposed to X-ray film for 2 days. F l y line are (1) 2M3, (2) 5 0 6 / 5 0 6 (3) +/+, (4) 2F2, (5) 6 M l a , (6) 6 M l b , (7) 6F2, (8) 4M1. The bands indicated are ( ) 9.2, ( ) 4.6, and ( ) 1.8 kb. 32  r  r  5  1 0 kb  - 4  I  '  H  h-  2 i  I H  R  II  7 J R  XDH 506  7 r 06 5  p Carnegie  Figure 12.  rosy  Locus of Drosophila  20  melanogaster  The rosy locus located on chromosome 3 at position 87 DE is represented here. The Xdh gene is shown as a rectangle with the 4 exons in black and the 3 introns in white. The position of the 3.4 kb r deletion is given, as is the Hind III fragment that was cloned into the P-element vector pCarnegie 20. Relevent restriction enzyme sites are shown. O kb is designated as the Eco RI site in exon 2. 506  48 water soluble molecules below 10 kV/cm.  Therefore, the reason for  the increased mortality of the larvae of embryos treated at high field strength is unknown. The presence of wild-type flies in the Fo generation could be the result of 3 different genetic events. 1. Somatic  transformation  of the embryos (Scavarda  Hartl, 1984; Maxwell and Maxwell 1988; Toneguzzo et al.  and  1986).  As  integration did not occur in the germline, this would cause expression of the Xdh  gene in the treated individual, but not in its progeny. 2.  Transformation  of  the  embryo  development such that some somatic Xdh  transformed  type eyes, and  gene.  and  very  germline  early  i n its  cells possess the  This would result in an Fo possessing  wild-  its progeny also being wild-type with respect to eye  colour. 3. The r °6/r 5  stock used for electroporation could  506  been contaminated with a wild-type fly at some point. few  of  the  heterozygotes untreated.  treated and  embryos  would  would  have  actually  have  Therefore been  r  5 0 6  a  / +  give Fo flies with wild-type eyes even i f  These flies would of course pass the wild-type gene onto  half on their progeny ( F i ) . As  wild-type Fo flies from control trials were also isolated, the  third possibility seems most likely. transformation showed  occurred  without  that transformants  could  One  pulsing. be  remote possibility is that Yoon  isolated by  and  Fox  simply  (1965)  incubating  premature embryos collected using an 'ovitron', in solution containing DNA  (Fox  and  Yoon,  1966).  A  small  percentage  of  such  49 embryos are permeable to water soluble molecules since the wax layer secretion does not occur i n embryos collected i n this way. However, as no transformants were detected  by Southern analysis,  this seems very unlikely. visual difference between rosy ~  The  is subtle, but distinct.  and wild-type eye  colour  The presence of even 1 % of the normal amount  of X D H has been reported to give wild-type eye colour (Chovnick et al,  1977).  A problem with this selection method is that the eye  colour does not fully developed until 1-2 days after eclosion. In addition, the desire to see wild-type eye colour when the difference is so slight, makes miss classification a problem. chemical  method  For these reasons, a  for distinguishing between rosy  " and wild-type  flies was utilized as a secondary screen. Purine  selection has been proven extremely useful for selection  of wild-type flies over rosy  "  mutants (Finnerty et al, 1970). The  0.2%  titre eliminated any leakage of the r  0.1%,  yet allowed heterozygotes to develop uneffected.  5 0 6  /r  5 0 6  that occurred at  this selection on the putative transformants eliminated phenotypically selected fly lines.  Utilization of half of the  However, as the amount of purine  required to k i l l the flies depends on the quantity of functional X D H expressed, it is possible that poorly expressing  transformants were  eliminated. Southern  analysis  of the purine  evidence for germline transformation.  selected  lines  showed no  A l l the bands derived  from  the putative transformants can be explained as coming from either a r  506  o r  wild-type Xdh  gene.  A s a result, one must conclude that  50 none  of the wild-type F i flies  pCarnegie 20 vector.  were i n fact transformed  by the  The most obvious explanation for the presence  of wild-type individuals in the Fn and F i generations is contamination of the r506/ 506 r  s t o c  k  w  i  m  and  a  w  t  fly.  C.  The Survival Embryos  Permeability  of  Dechorionated  1.  Variation i n the survival of dechorionated embryos Variation i n survival of untreated embryos was observed.  variability 13).  followed an approximately  However, the parameters  mode  of preparation  normal  distribution  The (figure  of the distribution differed with the  of the embryos.  dechorionated, mounted on tape, covered  r  506/ 506 r  with halocarbon  embryos o i l , and  placed i n a humid chamber, have great variability and a low mean survival (x=67%; s=12.5). In contrast, with r dechorionated  5 0 6  /r  5 0 6  and placed i n a humid chamber much less variation  was observed.  The standard deviation was half that above (s=5.9),  while the mean survival was 2 5 % higher (x=85%). only  spread  embryos simply  over  The data were  a 2 0 % margin (77-98%) compared to the 5 0 %  distribution (43-91%) for tape-mounted embryos.  Consequently, the  procedure of mounting embryos on tape was abandoned when it was found that addition of 0.05% TX-100 to the buffers would prevent the embryos from sticking to the sides of the cuvette and each other. 2.  Permeability of dechorionated  embryos  Incubation  embryos  of dechorionated  i n buffers containing  0.05% TX-100 and 0.01% Nile Blue revealed that these embryos were basically  impermeable to water  soluble macromolecules  such as  5 1  A.  o  Tape-mounted  A  Non-mounted  •  Mean  B.  10 n  ¥— —f— —¥—' 1 1 ' 1 —V~ 0-9 10-1920-2930-3940-4950-5960-6970-7980-8990-99 100 —"  V  V  1  1  V—•—¥—'  %  Figure  13.  1  1  1  Survival  Variability in the Survival of Dechorionated Embryos  506/ 506 flies (five to six days old) were placed i n a laying bottle i n the dark, and allowed to lay eggs for 2-3 hrs. Embryos were then collected at 25-35 minute intervals. The embryos were dechorionated, then treated i n one of two ways. Tape-mounted embryos were arranged side by side on double-sided tape i n groups of about 25-50, covered with halocarbon o i l and then placed in a humid chamber (dH20). Non-mounted embryos were placed directly i n a humid chamber (dH20) after having their chorion removed. The percentage of embryos to hatch within 48 hrs of collection is given. A. The points represent different collections on the same day, as well as collections from different days, and for the tape mounted embryos, different tapes from the same collection are also included. The mean for all trials is also given. B. The number of trials i n which the percentage survival was within the range indicated is graphed. r  r  52 dyes.  Less than 6 % of the treated embryos were able to take up  visible quantities of dye (data not shown). 3.  Discussion Dechorionated  Water  vapour  portions  embryos  remain  and respiratory  of the eggshell,  nucleotides, amino  essentially  gases  system.  can permeate the remaining  but even  acids, sugars  a closed  small  or water  molecules soluble  dyes  such  as  cannot.  However, the permeability of the dechorionated embryos to water vapour makes desiccation their primary cause of death. Removal of the chorion from Drosophila about 2-3 minutes. the  embryos takes only  However, the time required to properly mount  embryos on tape can be considerable (up to 15 minutes) and is  proportional to the number of embryos mounted and the dexterity of the  experimenter.  This difference i n the time that the vulnerable  embryos are exposed to the environment is significant. mounting  Although the  on tape takes place in a humid chamber (to increase the  local humidity), tape-mounted embryos still showed visible signs of desiccation after only a few minutes. completely  control  the environment  It was not common practice to surrounding  the specimens.  Several trials were performed i n a chamber of controlled humidity (75-85%) and temperature (17-18 °C); nevertheless, little difference in percentage survival was observed. and to  variation observed  Therefore, the poor  survival  between tape-mounted trials i n comparison  simply dechorionated embryos were likely due to differences i n  preparation time and the resulting extents of desiccation.  These  results also suggest that 8 0 % humidity was insufficient to prevent  53  significant desiccation. Due  to the variability  hard to draw conclusions small  differences  in survival between preparations from  numerical  i n survival.  values  However,  obtained,  it was or see  survival trends  were  reproducible and were somewhat informative. A  small percentage of dechorionated  to incorporate the blue dye.  embryos (< 6%) were able  One possible explanation for this is that  the wax layer is not impermeable in these embryos. observed  that  when  blowflies  were  induced  Davies (1947)  to l a y embryos  continuously, 5-10% of the embryos were osmotically sensitive.  This  suggested that the wax layer which is secreted just prior to laying had not solidified i n the embryos laid last. would  not possess  an intact  water  These embryos therefore  barrier and would  remain  permeable to water soluble molecules until the wax dried.  D.  Permeability  of  Embryos  Treated  with  TX-100  1.  Survival of embryos treated with increased concentrations of TX-100 Dechorionated  embryos  were  incubated  i n PBSuc  containing  increasing concentrations of TX-100 (figure 14). The results indicate that  survival was not remarkably  (below 0.5%) of TX-100.  After a 20 minute incubation i n buffer,  survival remained high (about 7 5 % ) . 100) could  A zero point control (no TX-  not be performed due to the adhesive nature  embryos in the absence of detergent. of  affected at low concentrations  untreated  dechorionated  of the  However, the average survival  embryos  was  85%  (see above).  54  Figure  14.  The Effect of T r i t o n - X 100 Concentration on E m b r y o Survival and Permeability to the Dye Nile Blue  506/ 506 f i i ( f i e to six days old) were placed in a laying bottle in the dark, and allowed to lay eggs for 3 hrs. Subsequently, dechorionated embryos from 30 min. collections were washed into a 0.2 cm cuvette with 500 p i of PBSuc containing Nile Blue (0.01% w/v) and varying amounts of T X 1 0 0 (v/v) . The cuvette was incubated at room temperature for 20 min. The embryos were then collected on Miracloth, rinsed with PBSuc, and placed in a humid chamber (PBSuc). The number of embryos collected after treatment (coloured and not) was recorded, as was the number of larvae which hatched within 48 hrs post treatment. r  r  e s  V  Increasing  the TX-100  concentration  to 5.0%  showed  a marked  decrease in percentage survival (to below 6 0 % ) . 2.  Permeability of embryos treated with increased  concentrations  of TX-100 Dechorionated Blue  and  various  embryos were incubated in PBSuc containing Nile concentrations  of TX-100.  Increasing  the  concentration of TX-100 had limited effect on embryo permeability during a 20 minute incubation (figure 14).  A t 5.0% TX-100, the  percentage of embryos able to take up the Nile Blue dye was still less than 10%. 3.  Discussion Due  to the low survival, great variation in survival, not to  mention laborious preparation of tape mounted embryos, a system for using freely suspended embryos was sought. have demonstrated that detergents concentrations, can be dechorionated  used  such as SDS  and TX-100 at low  to eliminate the 'stickiness' of the  embryos, yet did not interfere  development of the embryos.  Prior investigators  with  the  continued  In addition, detergents have also been  used to make the Drosophila  embryo permeable to radiolabeled  nucleotides (Eudy et al, 1969; Sayles et al, 1973). A  low concentration of TX-100 (0.05% (v/v)) was found to be  adequate for embryo  suspension.  The  embryos  could  easily  be  suspended i n buffer, and although they settled to the bottom of the cuvette due to gravity, they were readily dispersed and resuspended by  simply by taping the cuvette.  As previously stated, these non-  mounted embryos were much easier to prepare and use, and also  56  gave higher and less variable percentage survival values (85 /. 6 % ) . +  Incubation of the dechorionated  embryos i n buffer containing a  small amount of TX-100 (up to 0.5%) did not effect embryos survival. However, at higher concentrations (eg 5%) a noticeable increase i n This is i n agreement with Sayles et  mortality was apparent. (1973),  that Drosophila  who reported  Drosophila  embryos  Ringer's containing TX-100 developed  al.  incubated i n  normally providing  the detergent concentration did not exceed 0.5%, and the exposure was limited to 2 hours. A  20 minute incubation in the detergent containing buffer was  not sufficient to make the embryo permeable to the dye.  Sayles et al.  used at least a two hour incubation to achieve incorporation of [ H ] 3  amino  acids  or uridine  into  the embryo.  However, with the  constraint of transforming the pole cells (which form 1.5 hours after fertilization feasible.  at room  Decreasing  temperature),  a lengthy  the temperature  performed to 17 °C would extend  incubation was not  at which the experiment  was  the transformation window and  therefore permit a longer incubation.  However, a quicker method  would be preferred.  E.  Permeability  of  Embryos  treated  with  Solvents  1.  Survival of heptane-treated embryos incubated i n different buffers Survival of dechorionated embryos incubated in heptane for one  minute was extremely low.  When such embryos were incubated for  30 minutes i n any of the typical buffers, mortality ranged from 70-  57  100%  (figure  15).  Although the percent  survival varied between  trials, PBSuc gave the highest survival (18%).  In fact, over a number  of trials, 2 8 % survival (PBSuc) was the highest recorded for heptanetreated 2.  embryos.  Permeability  of heptane-treated  embryos incubated  i n different  buffers Heptane-treated embryos are extremely permeable to dyes such as  Nile  Blue  (figure  15).  80-95% of treated  embryos  coloured, regardless of the buffer used for the incubation.  became  This is a  18-19 fold increase in permeability as compared to controls ( 5 % ) . 3.  Survival of embryos treated with different solvents Dechorionated embryos incubated  for one minute i n a variety of  n-alkyl solvents showed a very poor ability to survive (figure 16). Following a thirty minute incubation in PBSuc containing 0.05% TX100 and 0.1% Nile Blue, survival was typically around 1 0 % regardless of the solvent used. 4.  Permeability of embryos treated with different solvents The three solvents were all able to increase the permeability of  the embryo to the Nile Blue dye (figure 16).  9 0 % or more of the  solvent-treated embryos were able to take up the dye. solvent  treatment  of Drosophila  embryos  permeable to the dye N i l e Blue, as well  made  as other  Although them  very  dyes such as  Toluidine Blue, Acridine Orange, and Methyl Red (data not shown), larger molecules were still excluded.  Figure 17 shows the results of  an incubation of solvent-treated embryos for 30 minutes i n PBSuc containing 0.05% TX-100 and 0.4% Blue Dextran.  No visible coloration  58  100 80 O)  5  c o  60-  I  % Permeable  E3  % Survival  a. 40-  20 0  PBSuc  Figure  PBSal  15.  DR Buffer  BIM  dH20  Survival and Permeability Treated Embryos  of  Heptane-  fij (five to six days old) were placed in a laying bottle in the dark, and allowed to lay eggs for 2 hrs. Subsequently, the egg laying plates were changed every 30 min. The embryos from each collection were dechorionated, rinsed with dH20, then rinsed off the Miracloth into a 100 mesh stainless steel cup using dH20. The embryos were then submerged i n d H 2 0 saturated heptane for 30 seconds and the excess solvent drawn off with paper towels. The embryos were rinsed with the buffer indicated , collected on Miracloth, then quickly washed off the Miracloth into a 0.2 cm cuvette with 500 p i of the indicated buffer. The buffers each also contained TX-100 (0.05% v/v) and Nile Blue (0.01% w/v). After a 30 min. incubation i n the solution, the embryos were collected on Miracloth and rinsed with the appropriate buffer (without detergent and dye). The cloth disk was laid on a glass slide, the embryos covered with halocarbon oil, and the slide placed in a humid chamber (indicated buffer). The number of embryos collected after treatment (coloured and not) was recorded, as was the number of larvae that hatched within 48 hrs of the treatment. r  506/ 506 r  e s  59  PENTANE  HEPTANE  DECANE  I  %  El  % Survival  Permeable  NO SOLVENT  Solvent  Figure 16.  The Effect of Different Solvents on E m b r y o Survival and Permeability the Dye Nile Blue.  to  5 0 6 / 5 0 6 flies (five to six days old) were placed in a laying bottle in the dark, and allowed to lay eggs for 2 hrs. Embryos were then collected at 30 min. intervals. The embryos were dechorionated rinsed using d H 2 0 and washed into a 100 mesh stainless steel cup with d t ^ O . The cup was then submerged into the indicated solvent for 30 seconds. The residual solvent was blotted away with paper towels. The embryos were then rinsed with PBSuc, collected on Miracloth, and washed into a 0.2 cm cuvette with PBSuc TN. The cuvette was incubated at room temperature for 20 min., at which time the embryos were collected on the cloth disk, rinsed with PBSuc and placed in a humid chamber (PBSuc). The number of embryos collected after treatment (coloured and not) was recorded, as was the number of larvae that hatched within 48 hrs of the treatment. No = embryos were incubated i n PBSuc instead of solvent. r  r  60  I  PENTANE  HEPTANE  DECANE  % Permeable % Survival  NO SOLVENT  Solvent  Figure  17.  The Effect of Different Solvents on E m b r y o Survival and Permeability Blue Dextran.  to  506/ 506 f i i (five to six days old) were placed in a laying bottle in the dark, and allowed to lay eggs for 2 hrs. Embryos were then collected at 30 min. intervals. The embryos were dechorionated rinsed using dH^O and washed into a 100 mesh stainless steel cup with dH20. The cup was then submerged into the indicated solvent for 30 seconds. The residual solvent was blotted away with paper towels. The embryos were then rinsed with PBSuc, collected on Miracloth, and washed into a 0.2 cm cuvette with PBSuc T X containing Blue Dextran (0.4% w/v). The cuvette was incubated at room temperature for 20 min., at which time the embryos were collected on the cloth disk, rinsed with PBSuc and placed i n a humid chamber (PBSuc). The number of embryos collected after treatment (coloured and not) was recorded, as was the number of larvae that hatched within 48 hrs of the treatment. No = embryos were incubated in PBSuc instead of solvent. r  r  e s  61  of any of the treated embryos was observed. 5.  Discussion The  of Drosophila  embryos  melanogaster  are generally  impermeable to water soluble metabolites, even upon removal of the chorion.  In 1973,  Drosophila  Limbourg and Zalokar reported that dechorionated  embryos  developed  normally,  radiolabeled  amino  briefly  submerged  yet the procedure acids.  equivalent to controls (95%)  Embryo  i n heptane  made them  or octane  permeable to  survival was reported  to be  when solvent treatment did not exceed  eight minutes. Unfortunately,  i n attempts to duplicate such  studies, solvent-  treated embryos did not survive to levels even closely resembling that of controls.  The high mortality rate could be due to one of two  things: residual solvent or excessive ranging  desiccation.  Different solvents  from pentane to decane were examined, thus investigating a  range of volatilities and water solubilities of the solvents. extremely during were  volatile  and therefore  subsequent procedures. washed  soluble removed  with  should  have  Pentane is  evaporated  quickly  Also, i n trials where the embryos  buffer, although  solvents  i n aqueous solutions, the extensive traces of the solvents.  are not extremely washing  would  have  However, the use of a l l three  solvents resulted i n extensive death of treated embryos.  Therefore,  the presence of residual solvent after treatment was not a major factor i n the death of solvent-treated embryos. Experiments conditions.  were  typically  not performed  under  humid  However, several trials were performed i n a room i n  62  which  the humidity was  these  experiments  held constant at 75-85%.  were  comparable  uncontrolled environment.  to  those  The  results of  performed  Therefore, either desiccation was  in  an  not the  reason for the increased death or more likely, the treated embryos are so vulnerable to desiccation that they require higher than  85%  humidity to avoid extensive dehydration. Although typically 8 0 % further  after  the  of the treated embryos failed to develop  solvent treatment, the procedure  did make  the  embryos extremely permeable to water  soluble dyes such  Blue.  the dead embryos that took  It could be suggested that it was  as N i l e  up the dye, however in controls (no solvent treatment) up to 2 5 %  of  the embryos failed to develop to the larval stage, yet only 0-6%  of  these treated embryos were able to take up the dye. solvent treatment was  removing  Therefore, the  some barrier (the waxy layer), thus  making the embryos permeable to water soluble substances. the small dyes are readily absorbed  While  by the solvent-treated embryos,  Blue dextran (MW  2 million) uptake was  not detected in any of the  treated embryos.  Since embryos remain  impermeable to the high  molecular  dextran, the  membrane  weight  intact, thus As  vitelline  still  be  preventing the uptake of large molecules.  the solvent treatment removes the waterproofing barrier of  the embryo, the nature of the buffer in which may  must  they are incubated  become important in terms of tolerance to the metabolites  present  and  cytoplasm Drosophila  the of  osmotic  the  difference  embryos.  Ringer's  between  Previous  the  buffer  investigators  and  the  have  used  solution or a cell culturing solution  (BIM  63  (Limbourg  and Zalokar,  1973)) for their  incubations,  yet these  buffers d i d not result i n an improvement i n the ability embryos to survive. medias  had little  mortality  of the  In fact, as figure 15 illustrates, the different  effect on embryo  was so extensive  survival.  However  since  (even for solvent-treated embryos not  subsequently incubated in buffer), the effect of the different buffers on the solvent-treated embryos cannot be assessed accurately.  F.  Effect  of  Temperature  on  Embryo  Permeability  1.  Survival of embryos at increasing temperatures Survival of dechorionated embryos incubated for 20 minutes i n  PBSuc containing TX-100 decreased with an increase i n incubation temperature (figure 18).  Survival decreased slightly  between room  temperature (22 °C) and 29 °C, then rapidly above this.  B y about 42  °C, few i f any embryos survived the incubation. 2.  Permeability of embryos at increasing temperatures The effect of temperature on the permeability of dechorionated  embryos was investigated. impermeable  A t low temperatures, the embryos were  to the N i l e Blue  temperature  was raised, their  dye, however, as the incubation permeability  increased  (figure 18).  Incubation in PBSuc containing 0.5% TX-100 and 0.01% Nile Blue for 20  minutes  had little  temperatures.  effect  on embryo  permeability  at l o w  A n increase in permeability was not observed  until  42 °C, where the percentage of embryos able to take up the dye jumped  from  Increasing  about  4%  (37 °C) to 4 5 % .  the temperature  A  further, resulted  dramatic increase. i n even  greater  64  100  Temperature  Figure  18.  (°C)  The Effect of Temperature on Embryo Survival and Permeability to the Dye Nile Blue  506/ 506 f i i (five to six days old) were placed in a laying bottle in the dark, and allowed to lay eggs for 2 hrs. Dechorionated embryos from 30 min. collections were washed into a 0.2 cm cuvette with 500 p i of PBSuc TN. The cuvette was placed in a water bath at the indicated temperature for 20 min.. The embryos were then collected on Miracloth, rinsed with PBSuc, and placed in a humid chamber (PBSuc). The number of embryos collected after treatment (coloured and not) was recorded, as was the number of larvae that hatched within 48 hrs of the treatment. r  r  e s  65  permeability.  B y 50 °C, almost 8 0 % of the treated embryos were  capable of dye uptake. 3.  Discussion In  1945, Wigglesworth  cuticle  of many  temperature.  insects  showed  that transpiration  increased  drastically  through the  above  a  Drosophila  In 1963, King and Koch showed that for  embryos which  were  already laid, incubation  critical  i n saturated  saline  solutions had no effect on survival unless the temperature of the solution was raised to 45 °C. A t this point, the embryos shrunk and as  a result,  demonstrated  failed  to develop  this i n blowfly  further.  Davies  embryos (critical  (1947) also  temperature  around  38 °C). These observations contributed to the hypothesis that the major waterproofing substance of such embryos was a wax layer. Figure  18 confirms that the above is true for  melanogaster.  The permeability  of the embryos  Drosophila  increased  dramatically between 37-42 °C, therefore suggesting that the critical temperature for melting the waxy layer was above 37 °C. The apparent  discrepancy  between  these  results  and the published  literature may be the result of the detergent present i n the buffer aided i n the extraction of the wax molecules, or simply that King and K o c h d i d not examine temperatures between room temperature and 45 °C. (They did not present any data over this range.) Unfortunately, at a temperature  at which  the embryos  quite permeable (42 °C), few survived the incubation.  were  Perhaps by  increasing the TX-100 concentration, or using another detergent with a lower  critical  micelle  concentration, the temperature  at which  66  permeability is observed could be lowered to a point where greater embryo survival makes the procedure viable.  G.  Conclusions  Exposure to an extreme field strength (10 kV/cm) was required to produce a  Drosophila  embryo which was permeable  melanogaster  to water soluble dyes.  However, such conditions were detrimental to  the  normal development  the  removal of the thin wax layer, without harming  must  first  be achieved  efficiently on achieved  of the embryos.  embryos.  Drosophila  by  three  before  methods:  It therefore appears that the embryos,  electroporation can  be  utilized  The removal of this layer can be  detergent  solubilization,  solvent  extraction and heating. Detergent solubilization of the wax did not occur quickly enough at  room  temperature to make the procedure feasible for use  transformation experiments. incubation  temperature,  transformation would wax  in  It is possible that by decreasing the the time  be extended.  available  f o r germline  However, this would  to become more rigid, thus requiring  cause the  either more detergent  (above 5.0% TX-100, which would be toxic to the embryo), or an even longer incubation to remove the barrier.  The temperature could also  be increased, thus combining heating and detergent solubilization to remove the wax.  However, this increase i n temperature  reduce the transformation window. little  effect  on increasing  would  Also the use of 0.5% TX-100 had  the permeability  of the embryo at  survivable temperatures, hence higher concentrations (perhaps 5.0%)  67  or alternate detergents would have to be investigated. Finding incubation  a  temperature  at this time. The  viable  combination and  of  detergent concentration,  transformation time  seems  improbable  Solvent extraction, on the other hand, seems promising.  technique is rapid, and may  even eliminate the need to remove  the embryos chorion.  A procedure which keeps the embryo in a very  humid,  environment  or  aqueous  throughout  the  procedure  still  remains to be found. Once the wax  is removed, the embryo should become more  receptive to the electrical current. (Assuming does not also interfere with the process.) to  achieve  pore  formation  decreasing  the  effects  important  factors  and  transformants.  of  would  The field strength required  presumably  electrophoresis  increasing  the vitelline membrane  the  decrease, of  probability  thereby  developmentally of recovering  68 IV.  REFERENCES  Andreason, G.L. and Evans, G.A. (1988) Introduction and expression of D N A molecules i n eukaryotic cells by electroporation. BioTechniques 6, 650-660. 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