Table Of Contenttì--g^î,6
THE ZEBRAFISH D'4,NIO RERIO;
A PISCINE MODEL FOR BIOTECHNOLOGY
by
Paul John Verma M.Sc.
Department of Obstetrics and Gynaecology,
University of Adelaide, Adelaide, Australia
A thesis submitted to the University of Adelaide in fulfilment of the
requirements for admission to the degree of Doctor of Philosophy
November t995
For Daphne, Lorraine and Ivan
II
TABLE OF CONTENTS
LIST OF TABLES AND FIGURES..... v11
LIST OF PLATES.. 1X
ABBREVIATIONS...... X1
ABSTRACT XlV
DECLARAÏON............. XV111
ACKNOWLEDGMENTS ... XlX
xx
PUBLICATIONS
INTRODUCTION .................'."" L
L.L
REVIEW.
LITERATURE ...,......,..2
1..2
MANIPULATIONS
L.3 SINGLE GENE ..........3
L.3.L Transgenic fish - The Pros and Cons... 4
l-.3.2 Gene Transfer Techniques Investigated 5
L.3.2.L Non piscean species....'..' 5
1..3.2.2 Pisceans 8
1.3.3 Gene Constructs Investigated............ 9
1.3.3.3 Future APPlications 11
1.3.4 Assessment of Transgenics ..'.' 12
1.3.4.tMosaicism """"""""72
1,.3.4.2 Lrtegration. 72
1..3.4.3 Expression. 13
1,.3.4.4 Germline transmissron ...'....'........' 13
PARTIAL GENOME MANIPULATION L3
1..4
1,.4.1 Embryonic Stem cells 1,4
1..4.2 Aggregation Chimeras .... 15
1,.4.3 Hybridization in fish 16
L.4.4 Ploidy Mosaic chimeras. 1.6
MANIPULATIONS...............
ENTIRE GENOME ....L7
1-.5
MANIPULATIONS
(A) CHROMOSOME SET ..........,..17
L.5.1 Triploidy ......... 18
1.5.1.1 Induction of TriPloidY 18
1.5.L.2 Survival and Viability of Triploids 20
1.5.2 Tetraploidy ........ 22
1..5.2.L hrduction of tetraploidy.'..........'..... 22
1,.5.2.2 Survival and Viability of Tetraploids 22
1..5.3 Analysis of Polyploidy'........'..'. ...................... 25
1.5.3.1 Karyotyping.................. 25
7.5.3.2 Erythrocyte (RBC) Nuclear Volume .'..... 25
1.5.3.3 Flow Cytometer......"....'. 26
1,.5.3.4 Coulter counter 26
1.5.3.5 Nuclear Components.....'............. 26
1,.5.3.6 Protein Electrophoresis .............' 26
t.5.3.7 Phenotypic Markers'....' 27
(B) MONOSEX POPULATIONS 27
L.5.4 Gynogenesis 27
1,.5.4.7 Sperm hractivation. 28
lll
Androgenesis """ 30
L.5.5
inactivation """""""""""30
1.5.5.1 Oocyie
I.5.5.2Phyiical treatments.. """"""""""31
L.5.6 Identification of Gynogenetic and Androgenetic Diploids..'.................31
L.5.6.1. Hybrid Markers """""""""""""'31
Markers...... """""""""""'32
1,.5.6.2Ailetic
Markers...... """"""""""'32
1.5.6.3 Genetic
1..5.7 SexControl of homozygous diPloid fish
1.5.8 Production of Inbred Lines of Fish 34
1.5.8.1 Inbreeding DePression 36
1.5.9 Sex Inversion.... 37
REARING.........
L.6 LARVAL """"'37
38
1.6.1 Feeds.........
1,.6.1,.1, Dry feeds... 38
1,.6.1,.2 Live feeds.. 39
1.6.1,.3 Frozen feeds........... 39
t.6.2 Physical Factors ...... 40
1..6.2.1, Water QualitY. "..'. 40
L.6.2.2 Temperature 41.
41.
1.6.3 Conclusion...'.. 41
.........
2.0 GENERAL MATERIALS AND METHODS .......43
..........43
2.L.L Hank's Balanced Salt Solution (HBSS) ..... ..........43
2.1,.2 F idn Ringer's Solution ..........43
2.1..3 Earle' s 199 Medium ..........43
2.1.4 Benzocaine (Ethyl-4-aminobenzoate) ......'.... 43
STOCKS
2.2 MAINTAINING FISH ...,..,.......44
2.2.1Danio rerio and D. frønkei 44
44
2.2.1.1. Tanks
47
2.2.2 Oryziøs løtipes
2.2.2.1. Tanks 47
2.2.2.2Food........... """""""'48
FISH
2.3 BREEDING ""' 48
2.3.1 Danios 48
2.3.L.1 Natural Spawning... 48
2.3.1,.2Artificial Insemination (AI) 49
2.3.2 Medaka. 50
2.3.2.1. Natural SPawning... 50
2.3.2.2 Artif icial Insemination 50
2.3.2.3In Vitro Maturation """"""""""'51
LARVAE
2.4 REARING OF DANIO .............51.
2.4.1Prcduction of Live Food... 51
2.4.1.7 Brine shrimp Artemia salina 51
2.4.7.2 Paramecia P. caudatum 52
2.4.1,.3 Microworms (Anguilla silusiae) 53
2.4.2 F eeding protocol.............'.. 53
2.4.2.1, First (Starter) Food.....'.. 53
2.4.2.2Secondary Live Food'.. 54
2.4.2.3 Supplemental DrY Food 54
lv
2.5 ANALYTICAL METHODS .............. 54
2.5.1, Production of transgene constructs... 54
2.5.2 DNA Analysis.. 55
2.5.2.L Collection of tissues.... 57
2.5.2.2 Purification of genomic DNA'..'..'.'.. 57
2.5.2.3 Polymerase Chain Analysis (PCR) 57
2.5.3 Transgene Expression Assays.. ,58
2.5.3.1 Collection of tissues.... ..58
2.5.3.2 Luciferase Assay 58
2.5.3.3 b-Galactosidase AssaY...' 59
2.5.4Karyotyping 59
3.0 SINGLE GENE MANIPULATION (TRANSGENESIS)........... 61-
INTRODUCTION 6L
3.1.
3.2. TRANSGENESIS METHODS........... 63
3.2.1 Micromanipulation for Gene Transfer 63
9.2.L.1, Micromanipulation System for Gene Transfer 63
3.2.7.2 Manipulation PiPettes 64
3.2.2 Trunsgene Preparation................ 64
3.2.3 Microinjection of DNA 65
3.2. BvaIuation of Gene Transfer 66
3.3. STATISTICAL ANALYSES............. 66
3.4 EXPERIMENTAL............... 66
3.4.L Comparison of Sperm Mediated Transfer of Circular and Linear
Constructs in the ZebrafishDanio rerio ................ 66
Outline....... """""""'66
3.4.L.7
g.4.1,.2 Results """""""""""67
3.4.2 Patterns of Expression of Luciferase, Exhibited by Zebrafish Embryos,
During Early DeveloPment.. 69
3.4.3 Inhibition of Parthenogenetic Activation of Ovulated Zebrafish
......70
Oocytes......
3.4.4Oocyte Injection as an Efficient Route to Producing Transgenic
Zebrafish........ 72
3.4.5 Sperm Mediated Transfer of Novel Genes in Medaka Oryziøs latipes..74
DISCUSSION....
3.s .......76
MANIPULATION........
4.0 PARTIAL GENOME .............. 94
4.1 INTRODUCTION 94
4.2 CHIMERIC METHODS 95
4.2.1- Fish Species..... 95
4.2.2 Supply of Embryos... 95
4.2.3 Micromanipulation for Production of Chimeras ... 95
4.2.3.1 Micromanipulation System .95
4.2.3.2 Manipulation Pipettes .'. .96
4.2.4 Melhodolo gy of Micromanipulation.......'....... 97
4.2.5 Manipulation media. 97
4.3 STATISTICAL ANALYSIS.............. 97
4.4 EXPERIMENTAL............... 98
4.4.1,Detection of Inter-Specific Chimeras of the Genus Danio, During Early
Development............ 98
Outline....... """""""'98
4.4.1,.1,
4.4.1,.2Results """""""""""99
4.4.2Conlribution of Transplanted Blastomeres in Adult Chimeras..........100
Ourline....... .............100
4.4.2.L
4.4.2.2Results """""""""" 100
4.4.3 Efficient Production of Inter-specific Chimeras using Cleavage
Donors
Advanced Embryos as ..............'..'.' 101
4'4'3'1' outline"""' """"""'101
4.4.3.2Results """""""""" 102
4.4.4Suwival and Subsequent Development of Manipulated Chimeric
embryos..
4.4.4.1. Outline.... ".
4.4.4.2Results ....................103
4.s DTSCUSSION.... ..... L04
5.0 INDUCTION OF TETRAPLOIDY IN THE ZEBRAFISH
RERIO .-.\24
DANIO
INTRODUCTION """""""""'124
5.L
METHODS...............
s.2 PLOIDY MANIPULATION ...................1.25
r25
5.2.L Heat Shock treatment.....
5.2.2 Analysis of ploidY 126
4N4LYSES............... .""'126
5.3 STATISTICAL
PROCEDURES...
s.4 EXPERIMENTAL .......T27
5.4.1, P ar ameters for temperature induced tetraploid y in zebt afish .... -. -. - -.. 127
Outline""." """"""'127
5.4.1..1.
5.4.1.2Results """"""""""127
tetraploidy......
5.4.2Cleavage rate as a gauge of incidence of ....L28
S. .Z.1 Outline....... .............128
5.4.2.2Results """"""""""L29
...........
5.4.3 Maximising survival of tetraploids """"" 130
Outline....... .............130
5.4.g.1.
Results ....................13L
5.4.3.2
5.4.4 Survival and viability of Tetraploids.......... 131
5.4.4.1. Outline....... ......................... 131
5.4.4.2Results """""""""" 131
DISCUSSION.... .....132
s.s
DISCUSSION .I44
6.0 GENERAL
BIBLIOGRAPHY L50
vl
LIST OF TABLES AND FIGURES
vector......
Figure 2.1The organization of the pGl2-control ..................56
embryos..
Table 3.1 Sperm mediated transgenesis in zebrafish .........8L
Table 3.2 Transgenesis in F0 and Fl zebrafish resulting from sperm mediated
transfer... """""""""82
Table 3.3 Sequential expression of luciferase by zebrafish embryos.................83
Table 3.4 Effect of sperm concentration on inhibition of parthenogenetic
activation of oocytes 84
Table 3.5 Fertilisability normality and survival of embryos following
transgenes...........
microinjection of .......85
Table 3.6 Comparative levels of expression of pGL2 injected into either
2ygotes...............
oocytes or .....................86
Table 3.7 Incidence of transgenesis in F0 and FL generations resulting from
iniection...............
zygote and oocyte .....87
Table 3.8 Introduction of transgenes into medaka embryos; survival and
rate....
transgenesis ..................88
Figure 3.L Sequential expression of luciferase by zebrafish embryos..................89
Figure 3.2Inhibition of oocyte activation; effect of sperm concentration on
fertilisability.....
subsequent ..----............90
Figure 3.3 Luciferase expression by embryos injected at either the oocyte or
zygote stage. 9r
vll
Table 4.1 Embryo survival and frequency of pigmented chimeras following
transfer
blastomere """""""'107
Table 4.2 Survival to maturity of inter-specific chimeric embryos....................108
Table 4.3 Production of chimeras resulting from the use of advanced embryos
as a source donor blastomeres............ L09
Table 4.4 Effect of donor cell-number on survival of chimeras.........................11'0
Table 4.5 Comparison of overall survival and deformity observed in embryos
groups.....
from different treatment ""'111
Table 5.1 Survival of zebrafish embryos following heat shock (HS)................136
zebrafish..........
Table 5.2 Induction of tetraploidy in """"137
Table 5.3 Frequency of tetraploid zebrafish in normal and abnormal embryos
treatment...........
following HS """"""'L38
status..
Table 5.4 Cleavage rate as an indicator of tetraploid .............139
Table 5.5 Effect of duration of HS on induction and normal survival of
tetraploids............... ."""""""L40
tetraploidy.........
Figure 5.1 Normal survival a/s induced ...141
zebrafish
Figure 5.2 Survival of tetraploid """""'142
vlll
LIST OF PLATES
Plate 1.1 Species of fish used for this study xxll
plate 3.L Detection of the pGL2 transgene in fins of adult putative transgenic
zebrafish ..,..,.....92
latipes
Plate 3.2 Sperm mediated gene transfer in medaka o. .........93
Plate 4.1 Removal of blastomeres from donor embryos 112
Plate 4.2 lnjection of donor blastomeres into recipient embryos................... LL3
Plate 4.3 Chimeric manipulations: Control embryos at 36h of developmentLL4
Plate 4.4 Chimeric manipulations: Chimeric embryo at 36h 1.1.5
Plate 4.5 Chimeric manipulations: Control embryos at 48h 116
Plate 4.6 Chimeric manipulations: Chimeric embryos at 48h........,..,...,.""""117
Plate 4.7 Chimeric manipulations: Control embryos at 55h LL8
Plate 4.8 Chimeric manipulations: chimeric embryos at 55h......................... L19
Plate 4.9 Chimeric manipulations: Control adult fish 120
Plate 4.L0 Chimeric manipulations: LD-ZD adult chimera L (male) .............121
Plate 4.L1 Chimeric manipulations: LD-ZD adult chimera 2 (female).........,122
IX
Plate 4.L2 Chimeric manipulations: LD'ZD adult chimera 3 (male) 123
Plate 5.1. Zebrafish embryos resulting after HS induced tetraploidy............t43
x
Description:THE ZEBRAFISH D'4,NIO RERIO;. A PISCINE MODEL FOR BIOTECHNOLOGY by. Paul John Verma M.Sc. Department of Obstetrics and Gynaecology,. University of Adelaide, Adelaide, Australia. A thesis submitted to the University of Adelaide in fulfilment of the requirements for admission to the degree
