GENE TRANSFER IN FISH

1
GENE TRANSFER IN FISH

• Incorporation of foreign DNA (transgene) into
  fish genome
• Methods of gene transfer
• Applications in aquaculture and environmental
  science

2
STAGES OF GENE TRANSFER

• Cloning of transgene
• Transformation delivery of DNA
• Verification of transfer and expression of
  transgene
• Generation of germ-line transgenic fish
• Analyses of incorporation, expression and
  phenotypic effects

3
GENERIC TRANSGENE
cDNA
promoter
coding sequence (cDNA)
terminator
Minigene
promoter
cDNA
terminator
enhancer
intron
Full-length-gene

4
PROMOTERS

• Mammalian/avian viruses
• strong, constitutive, all-tissue
• RSV (Rous sarcoma virus)
• TK (thymidine kinase)
• SV40 (simian virus)

• CMV (cytomegalovirus)

5
Reporter genes detection

• Luciferase luminiscence assay
• CAT radioactive assay
• LacZ colorimetric assay/staining
• GFP fluorescence microscopy

6
Reporter genes applications
Tissue distribution
In vivo selection
Quantification
Sensitivity
CAT -
- Luciferase
- LacZ
GFP

7
SALMONID PROMOTERS

• Histon 3
• Metallothionein A and B
• Protamin
• Hepatocyte nuclear factors -1 and -3
• Mx1
• Vasa-like gene
• Cardiac peptide hormone
• Insulin, insulin-like growth factor II
• Growth hormone, prolactin, gonadotropin,
  gonadotropin-releasing hormone, Pit-1
• Albumin, tranferrin
• Estrogen receptor
• Heat-shock-protein-like gene

8
FISH PROMOTERS

• Tissue-specific/all-tissue
• Constitutive/inducible
• Strong/weak
• Broad/narrow host specificity

9
Tissue-specificity regulatory elements
enhancer
promoter
gene
100-300bp
2500-17000 bp
10
Muscle-specific expression of skeletal
aSkActin-GFP in zebrafish
Higashijima et al., Dev. Biol.,1997
11
Expression of skeletal aActin-LacZ in rainbow
trout embryo
12
Transfection model objects

• Established cell cultures
• Embryos Primary cell cultures

13
Delivery of DNA microinjection
14
Delivery of DNA microinjection

• Developmental stage 1-8 cells
• Processing of chorion
• Transgene copies 104-106

Transformation rate
Survival
Transgene copies
15
High-throughput methods

• Electroporation
• Sperm-mediated DNA loading or electroporation
• Lipofection
• Particle bombardment (biolistic)

16
Transgenes in fish

• Selection of positive individuals
• PCR analyses of fin clips
• Transgene integration
• Southern blots, linker-assisted PCR
• Transgene expression
• Northern blots, RT PCR
• Inheritance

17
Problems...

• Low yield of transgenics
• Lack of integration or
• Integration into silent sites
• Lack of expression in F0 or F1
• Poor inheritance

18
Solutions for improved integration and expression

• Additives (peptides)
• Viral NLS peptide
• Improved integration into active sites of
  chromatin
• Enhanced expression and inheritance
• MoMLV integration protein
• Improved integration

19
Novel vectors transposons
Artificial Tc1/mariner-like transposon
transposase
Sleeping Beauty
CMV
transposase
transgene
20
Active transposon (Tol2) was found in medaka
21
Novel vectors

• Artificial chromosomes
• BAC
• YAC
• Retroviral
• MoMLV virus
• Envelope protein of vesicular stomatitis virus

22
Embryonic stem (ES) cells
23
Zebrafish ES cells (Ma et al., 2001
Differentiation into germ-line cells? Passage?
24
Nuclear transfer zebrafish
25
Nuclear transfer zebrafish
26
Nuclear transfer zebrafish
27
Applications of gene transfer

• Aquaculture
• Improved performance of fish
• Environmental monitoring
• Control of pollution
• Basic research
• Functions of genes in development

28
Growth enhancement milestones
hGH-transgenic goldfish Transfer of mammalian GH
in aquaculture species
1985
1990
Development of all-fish constructs dramatic
acceleration of salmonid growth Germ-line
transgenic stocks
Studies of phenotypic effects of transgenes
2001
29
GH-transgene effect on growth

• Modest (10-50)
• Goldfish, carp, loach, channel catfish, Northern
  pike, rainbow trout
• Moderate (100-200)
• Tilapia
• Dramatic (to 1500)
• Salmo, Salvelinus, Oncorhynchus

30
GH-transgene constructs
Arctic charr, 8 months
Growth effect frequency of malformations CMVgtMTgt
H3
CMVonGH1

onMTGH1
onH3GH1
ssGH2
control
31
Metabolism of GH-transgenic charrgas exchange
32
Metabolism of GH-transgenic charrplasma
metabolites
triglycerides
cholesterol
Time after feeding (h)
33
Metabolism of GH-transgenic charrplasma
metabolites
CO2
NH4
Time after feeding (h)
34
Metabolic alterations in GH-transgenic charr

• Reduced expenditure of dietary protein
• low ammonia to oxygen ratio
• high ammonia to carbon dioxide ratio
• Increased utilization of dietary fat
• rapid clearance of triglycerides
• no alteration in cholesterol levels

35
CELLULAR MECHANISMS OF GROWTH

• Tissues grow due to
• cell division (hyperplasia) - indefinite growth
• increase of cell size (hypertrophy) - limited
  growth
• In skeletal muscle these processes are separated
  between
• myoblasts (hyperplasia)
• myotubes (hypertrophy)
• Number of small myofibers is greater in rapidly
  growing fish

36
GH-transgenic Arctic charr cellular growth of
skeletal muscle

• What process was stimulated?
• proliferation of myoblasts?
• growth of myotubes?
• Relationship between specific growth rate (SGR)
  and body weight (W)
• Control charr SGR W -0.35
• Transgenic charr SGR W -0.3

37
GH-transgenic charr cell composition of muscle
Total number of white muscle fibre
Complete transverse section
38
Dr. Muir and Troyan genes
39
'Frankenfish' spawn controversy Debate over
genetically altered salmon Jane Kay, Chronicle
Environment Writer
40
Threats from Troyan gene

• (1) transgene increases male mating success but
  reduces adult viability,
• (2) transgene increases viability but reduces
  male fertility, and
• (3) transgene increases both male mating success
  and adult viability but reduces male fertility.

41
Gain of new traits

• Cold tolerance
• Anti-freeze proteins
• Modification of metabolism (metabolic
  engineering)
• Gulonolactone oxidase - L-ascorbic acid
  biosynthesis
• Glucose transporter, hexokinase - utilisation of
  carbohydrates
• NO SUCCESS SO FAR

42
Gain of new traits resistance to diseases

• Non-specific
• Interferon
• Lysozyme
• To pathogens
• Silk worm cecropin (small proteins that act
  against pathogenic bacteria)

43
CECROPIN FIRST RESULTS

• Medaka
• Pseudomonas fluorescens and Vibrio anguillarum
  killed 40 of control and 10 of transgenic fish
• Channel catfish
• Flavobacterium columnare killed 73 of control
  fish, transgenics survived

44
Loss of function permanent

• Reproduction control in Tilapia by inactivation
  of GnRH
• cloning of GnRH promoter
• anti-GnRH ribozyme
• anti-GnRH cDNA
• Decrease of pituitary GnRH level
• no effect on reproduction
• dsRNA-dependent protein kinase (PKR) and
  2-5,-oligoadenylate synthetase

45
Loss of function temporary

• Morpholinos oligonucleotides
• phenocopies of mutations in zebrafish,
  inactivation of GFP expression
• dsRNA oligonucleotides
• inactivation of GFP expression
• Ribozymes
• codelivery of ribozyme, T7 autogene vector and
  T7 RNA polymerase

46
RNA interference
47
siRNA inhibit expression of GFP and TK in rainbow
trout embryos
48
Control of pollution
Induction
Expression
Promoter with RE
Luciferase

• Responsive elements (RE)
• Electrophile (quinones oxidants)
• Hydrocarbon
• Metal (Hg, Cu, Ni, Cd, Zn)
• Estrogen
• Retinoic