Characterization of zebrafish vitellogenin gene family for potential development of receptor mediated gene transfer method 1

Chapter 1. General Introduction


Chapter 1.
General Introduction

1
Chapter 1. General Introduction
1.1. Oogenesis and vitellogenesis
1.1.1. Oogenesis
The development of an egg is known as oogenesis, which can be divided into different
phases including 1) proliferation of primordial germ cells, 2) mitotic division of oogonia
within ovary, 3) progress through the early stage of meiosis and arrest in the prophase of
meiosis I (primary oocyte), 4) further growth and development of the primary oocyte, 5)
completion of division of meiosis I and arrest at metaphase of meiosis II (secondary
oocyte), 6) completion of meiosis II (egg) before or after ovulation depending on species
(Wolpert et al., 1998).

Eggs are filled with maternally provided building blocks (mostly RNAs and proteins) for
the developing embryos and these materials are incorporated into the oocytes during the
meiotic arrest stage before initiation of the division of meiosis I. In particular, eggs of
oviparous (egg-laying) animals, unlike those of mammals, accumulate enormous amounts
of proteins, lipids, glycogen (collectively called yolk) during this meiotic arrest period in
order to support future embryonic development (Kalthoff, 1996). Precursors of the major
yolk protein components are called vitellogenins (Vtgs), which are synthesized in the liver
of oviparous vertebrates or in the fat body of most insects under the control of hormones,
released into the blood circulation or hemolymph, taken up by oocytes through receptor-
mediated endocytosis and further processed inside oocytes (Wahli, 1988). The whole
process from Vtg synthesis to the sequestration of processed Vtg products in the oocytes is
termed vitellogenesis (Kalthoff, 1996).

2
Chapter 1. General Introduction
In all teleosts studied to date, oocytes undergo the same basic pattern of growth (Tyler and
Sumpter, 1996). Based on morphological criteria and physiological/biochemical events,
the oocyte development in the zebrafish (Danio rerio) can be divided into five stages
(Selman et al., 1993). In stage I (primary growth stage), an oocyte resides in the nest with
other oocytes (stage IA), and then is surrounded by a single layer of follicle cells followed
by continuous growth before arrest in diplotene of the meiotic division I (stage IB). In
stage II (cortical alveolus stage) and III (vitellogenesis), oocytes are distinguished by the
appearance of cortical alveoli and yolk proteins, respectively. In stage IV (oocyte
maturation), meiosis is reinitiated and the nucleus (germinal vesicle) migrates towards the
oocyte periphery. After ovulation, a mature egg is formed (stage V). During the primary
growth stage, an acellular vitelline envelop (referred to as zona pellucida, zona radiata or
chorion vitelline envelop) develops around the oocyte and continues to differentiate and
increase in complexity throughout the remainder of oocytes growth (Tyler and Sumpter,
1996). Since the early developmental stages of many species of fish last long periods of
starvation before first exogenous feeding, the maternal production of Vtgs and the
deposition of adequate supplies of yolk are essential to the survival of fish embryos and
larvae.

1.1.2. Vitellogenesis
The concept of hormonal control of vitellogenesis was first proposed by Bailey (1957)
after studying the correlation of estradiol (E
2
) with blood Vtg content in goldfish. The
concept has been tested and modified after extensive studies of vitellogenesis in insects
and amphibians. The hormonal control of vitellogenesis in frogs and insects is similar

3

Chapter 1. General Introduction
A
B
Fig. 1-1. Hormonal control of vitellogenesis and basic structure of vitellogenin proteins.
A: Hormonal control of vitellogenesis in frogs and insects (from Kalthoff, 1996). B:
Three basic Vtg organization schemes from three different phyla (from LaFleur, 1999).
See text for detailed descriptions.

4
Chapter 1. General Introduction
(schematically shown in Fig. 1-1A; from Kalthoff, 1996). Briefly, in amphibians,
environmental cues stimulate the hypothalamus to secrete gonadotropin-releasing
hormones which cause the pituitary gland to produce gonadotropins. These hormones
stimulate the ovarian follicle cells to produce estrogen, which in turn cause the liver to
synthesize Vtgs. In insects, the corpora allata (functionally analogous to the pituitary
gland in amphibians) release juvenile hormone which stimulates the ovarian follicle cells
in dipterans to produce a steroid hormone, ecdysone. The dipteran juvenile hormone or
ecdysone stimulates the fat body (functionally analogous to the liver in amphibians) as
well as follicle cells in to produce yolk protein precursors (Kalthoff, 1996). Vtgs or yolk
protein precursors are secreted into the blood stream or hemolymph and then sequestered
in the oocytes by receptor-mediated uptake. In Xenopus, Vtgs are further broken down
into lipovitellins (LVs) (I and II) and phosvitin (PV) or two phosvettes (due to an
additional cleavage in the PV domain) after uptake into oocytes (Wiley and Wallace,
1981). Cathepsin D was found to be responsible for the cleavage at the C-terminal end of
phosvitin (Opresko and Karpf, 1987).

Vitellogenesis is a major event responsible for the dramatic growth of oocytes in many
teleosts and may account for 11-95% of the final egg size (Tyler and Sumpter, 1996). The
duration of vitellogenic phase and the minimum size of oocytes before entering
vitellogenic development vary in different fish species. In most fish studied, hepatically-

derived Vtgs are the principle precursors of yolk proteins and their synthesis in the liver is
in response to the circulating E
2
derived from the ovary (Ng and Idler, 1983; Tyler, 1991).
Like other oviparous vertebrates, circulating Vtgs are selectively taken up by oocytes
through receptor-mediated endocytosis (Chan et al., 1991; Tyler and Lancaster, 1993).

5
Chapter 1. General Introduction
Studies indicated that the ability of an oocyte to sequester Vtgs depends on the
development of patency or opening of intercellular channels, which allows Vtgs to pass
through the follicular tissues to the oocyte surface (Wallace, 1985; Tyler et al., 1991). The
presence of hormone and growth factor regulated Vtg receptors on the oocyte surface is
also a key factor affecting vitellogenic growth of fish oocytes. Hiramatsu et al. (2002)
demonstrated that the LV domain of white perch (Morone americana) Vtg mediates its
binding to the oocyte receptor and the remaining domains may interact with the LV
domain to facilitate the receptor binding. Li et al. (2003) further narrowed down the
receptor-binding region in tilapia (Oreochromis aureus) Vtg1 to an N-terminal 85-amino
acid fragment in LVI domain.

After uptake into oocytes, Vtgs in teleosts, like those in amphibians, also undergo
proteolytic cleavage to form three main products, LVI (heavy chain), PV and LVII (light
chain) (Sharrock et al., 1992; Matsubara et al., 1999; Fig. 1-1B). Additional proteolytic
cleavages were observed either in LVI for lamprey Vtg (Sharrock et al., 1992) or in LVII
for Vtgs of several other fish species, resulting in a C-terminal β-component of ~260
amino acids (Matsubara et al., 1995; Hiramatsu and Hara, 1996). One unique feature in
fishes that produce floating eggs is that there is a second proteolytic cleavage event during
oocyte maturation and hydration which causes an increase of free amino acid content and
generates osmotic effectors needed for water influx (Carnevali et al., 1999). It has been
shown in seabream (Sparus aurata) that cathepsin D and B are involved in the first

proteolytic cleavage and cathepsin L is responsible for the second proteolytic cleavage,
resulting in complete degradation of one LV component (Carnevali et al., 1999).


6
Chapter 1. General Introduction
A variety of changes have been observed in hepatocytes of vitellogenic females or
estrogen administrated male fish and these changes are consistent with substantial
increases in the capacity of protein synthesis and export in the liver. Briefly, vitellogenic
hepatocytes are characterized by expanded nuclear envelope cisternae, swollen
mitochondria, enhanced rough endoplasmic reticulum, Golgi apparatus and secretory
vesicles, increased contents of proteins and total RNAs and increased amount of enzymes
such as transaminases and those of the Krebs cycle and glycolysis (Mommsen and Walsh,
1988 and references within). The de novo synthesis of vtg mRNAs and increased amount
of rRNAs may account for the increase in total RNA, and enhanced translational activities
are expected from the proliferation of translational machineries. Thus, E
2
is able to
orchestrate cell metabolism and biosynthetic activities at various levels in the liver of
teleost fish (Mommsen and Walsh, 1988).

1.2. Vitellogenin
1.2.1. Vitellogenin proteins
The term “vitellogenin” was first used to refer to a serum form of a yolk protein precursor
isolated from the Cecropia moth (Pan et al., 1969). Now, the term vitellogenin has been
reserved for yolk protein precursors that belong to an ancient gene family existing in a
wide range of metazoans from nematodes to insects and vertebrates (LaFleur, 1999).

Vitellogenins produced by oviparous vertebrates are large lipophosphoglycoproteins,
which are extensively modified with covalently linked carbohydrates, phosphates and

sulfates and with noncovalently bound lipids, hormones, vitamins and metals (Chen et al.,
1997 and references within). Native Vtgs in the blood circulation of most oviparous

7
Chapter 1. General Introduction
vertebrates studied so far are in the form of dimers with molecular weights between 326-
550 kDa, which are composed of Vtg subunits of 140-220 kDa (Mommsen and Walsh,
1988; Byrne et al., 1989). The three major yolk proteins (LVI, LVII and PV) found in the
eggs of oviparous vertebrates can be easily recognized as domains along the primary
structures of vtg cDNA translations. At the N-terminal of Vtg lies a relatively large
domain representing yolk protein LVI. At the C-terminal there is a relatively small domain
representing yolk protein LVII and the middle polyserine domain represents yolk protein
PV (LaFleur, 1999; Fig. 1-1B). It was suggested that the PV domain has undergone both
contraction and expansion from low to high vertebrates (Bidwell and Carlson, 1995).
Production, secretion and cleavage of Vtg precursors in different phyla of oviparous
invertebrates are more heterogeneous than in oviparous vertebrates. The native Vtgs in
invertebrates is either in the form of monomer (170-195 kDa) or dimer (530-550 kDa),
with or without posttranslational modifications and with (before or after uptake into
oocytes) or without cleavage when forming the yolk (Wahli, 1988; Byrne et al., 1989; Fig.
1-1B).

The most apparent difference between vertebrate and invertebrate Vtgs is that the
invertebrate Vtgs lack the polyserine-rich phosvitin domain (Spieth et al., 1985; Nardelli
et al., 1987; Fig. 1-1B). Interestingly, intact or vestigial polyserine domains were
identified in Vtgs of several insect species, including boll weevil (Anthonomus grandis),
mosquito (Aedes aegypti) and silkmoth (Bombyx mori) (Trewitt et al., 1992; Chen et al.,
1994a; Yano et al., 1994). Further analysis indicated that the polyserine domains of
arthropod and vertebrate Vtgs are unlikely to have originated from a common ancestor,
since not only are the locations of these polyserine domains different, but also the usage of

8
Chapter 1. General Introduction
serine codon differs between Vtgs in insects (mainly by TCX serine codons) and
vertebrates (mainly by AGY serine codons) (Chen et al., 1997; LaFleur, 1999). The
polyserine domains are heavily phosphorylated and may be important in maintaining
tertiary structure of Vtgs or in carrying calcium phosphate in support of vertebrate
embryonic bone formation (Wahli, 1988).

Although the divergence at the amino acid level is high, Chen et al. (1997) have reported
that there are five relatively conserved regions widespread along the Vtgs from
nematodes, insects and vertebrates. These five homologous subdomains (I-V) are located
outside both the polyserine domains of insect Vtgs and the phosvitin domain of vertebrate
Vtgs, and are aligned relatively easily among different phyla (Fig. 1-1B). Further
confirmation of the homology of Vtgs came from the observation that glycine, proline and
cysteine, which are important in secondary structure, were the predominant residues
among the strictly conserved residues (Chen et al., 1997). Thus, sequence analysis
supports that Vtgs of nematodes, insects and vertebrates share common ancient ancestry
(Chen et al., 1997).

Combining secondary structure features such as α-helixes and β-sheets, Babin et al. (1999)
further identified twenty-two N-terminal conserved sequence motifs (N1 to N22) covering
the homologous subdomains I-III and seven C-terminal conserved motifs (C1 to C7)
covering subdomains IV and V in invertebrate and vertebrate Vtgs. Interestingly, the
conserved motifs N1 to N22 were also identified in the N-terminal part of several
nonexchangeable apolipoproteins, including insect apolipophorin II/I precursor (apoLp-
II/I), human apolipoprotein B (apoB) and the large subunit of mammalian microsomal

9
Chapter 1. General Introduction
triglyceride transfer protein (MTP), suggesting a derivation from a common ancestral

functional unit, termed large lipid transfer (LLT) module (Babin et al., 1999).
Furthermore, the seven conserved sequence motifs (C1 to C7) were also identified in the
C-terminals of insect apoLp-II/I and human apoB (reminiscent sequence motifs), and
named as the von Willebrand factor D (VWD) module previously characterized in von
Willebrand factor (VWF) and several other proteins (Babin et al., 1999). In addition, there
are four and one conserved ancestral exon boundaries in the LLT and VWD modules,
respectively. Thus, the same authors also concluded that genes coding for Vtg, apoLp-II/I,
apoB and MTP large subunit were derived from a common ancestor and were members of
the same multigene superfamily, named as large lipid transfer protein (LLTP) superfamily
(Babin et al., 1999). Phylogenetic analysis also indicated that insect apoLp-II/I and
mammalian apoB are paralogous to Vtgs (Babin et al., 1999). As a matter of fact, apoB
and apoLp-II/I are implicated in the deposition of yolk reserves in birds and insects (Evans
and Burley, 1987; Soulages and Wells, 1994).

1.2.2. Vitellogenin genes
Vitellogenin (vtg) genes constitute a multigene family in most oviparous animals. For
example, six vtg genes have been identified in the nematode (Caenorhabditis elegans),
two in the migratory locust (Locusta migratoria), four in Xenopus laevis and three in
chicken (Gallua gallus) (Spieth et al., 1991; Locke et al., 1987; Schubiger and Wahli,
1986; Wang et al., 1983). In teleost, vtg mRNA sequences derived from more than one vtg
have been reported in GenBank for many species, indicating that teleost genomes also
contain multiple members of vtg genes.


10
Chapter 1. General Introduction
The multiple copies of vtgs are located either on one chromosome, such as in L.
migratoria and rainbow trout (Oncorhynchus mykiss) or on two chromosomes, such as in
C. elegans and X. laevis (Bradfield and Wyatt, 1983; Trichet et al., 2000; Spieth et al.,
1991; Wahli, 1988). Interestingly, most vtgs in nematode and the two vtgs in migratory

locust are located on the sex chromosome X (Spieth et al., 1991; Bradfield and Wyatt,
1983). Based on the genome organization of vtgs and sequence similarity, among different
vtg members, it seems that vtgs in different species may have undergone gene duplication
during evolution. For example, the nematode vit-3 and vit-4 may arise from tandem
duplication of a precursor gene, since both genes are tandemly linked in a head-to-tail
fashion and share high percentage of sequence identity (>99%) (Heine and Blumenthal,
1986; Wahli, 1988). In X. laevis, it was speculated that after an early duplication of a
primordial vtg, a more recent whole genome duplication took place, resulting in four vtgs
(A1, A2, B1 and B2) in modern X. laevis (Jaggi et al., 1982). Buisine et al. (2002)
postulated that the rainbow trout vtgs might be subjected to several rounds of
amplification, including an initial tandem amplification of precursor vtgs, resulting in ~10
copies of vtg genes and pseudogenes, followed by amplification of the whole vtg cluster,
leading to a three-fold increase in the total vtg copy number.

Although the length of the coding sequence is similar, the intron number and the average
intron size of vtg increase from the nematode to vertebrates, resulting in considerable
variation in the length of vtg genes. For example, the nematode vtgs have 4-5 exons, while
35 exons are present in vtgs of Xenopus and chicken, and 34 exons in teleost vtgs due to
the merging of the exons corresponding to exons 22 and 23 of the Xenopus and chicken. It
is not clear whether the amphibians and birds split this domain by intron insertion after

11
Chapter 1. General Introduction
radiation from fish or the teleost eliminated one of the introns during evolution (Mouchel
et al., 1997).

1.2.3. Regulation of vtg expression
1.2.3.1. Tissue specific expression of vtgs
The hormonal regulation of vitellogenesis and the site for vitellogenin (Vtg) synthesis
differ in invertebrates from oviparous vertebrates. In nematodes, Vtgs are synthesized in

the intestine of hermaphrodites (Kimble and Sharrock, 1983); whereas in echinoderms
such as sea urchin, Vtgs are synthesized in the intestine as well as in the gonads of both
sexes (Shyu et al., 1986). In insects, the synthesis of yolk proteins is mainly controlled by
juvenile hormone and also by ecdysteroids in Diptera (Wyatt, G.R., 1988; Byrne et al.,
1989). Unlike the vertebrate system, female-specific synthesis of yolk proteins in insects
is also controlled by products of sexual differentiation genes (Belote et al., 1985). In terms
of the expression sites, Vtgs or yolk proteins are synthesized in the fat body of female
insects or in both female fat body and ovarian follicle cells in the higher Diptera (Wahli,
1988). In oviparous vertebrates, Vtgs are synthesized mainly in the liver of female animals
and under the control of estrogen (Byrne et al., 1989 and references within). In addition,
different components of the ovarian follicle (somatic tissues surrounding an oocyte,
including granulosa, theca and surface epithelium) were also implicated in the
contribution of vitellogenesis in amphibians (Wallace, 1985) and squamate reptiles
(Andreuccetti, 1992). Evidence from a recent report indicated that the ovarian follicle cells
in a cartilaginous fish the spotted ray (Torpedo marmorata) synthesize Vtgs (Prisco et al.,
2004).


12
Chapter 1. General Introduction
1.2.3.2. Estrogen-dependent expression of vtgs in oviparous vertebrates
Vertebrate Vtgs are synthesized in hepatic parenchymal cells under the influence of
estrogen. Estrogen enters the liver and binds to the estrogen receptor (ER), and the
hormone-bound receptor binds tightly at the estrogen-responsive element (ERE) located
upstream of, or within, estrogen-responsive genes such as ER and vtg, resulting in
activation or enhanced transcription of these genes (Lazier and MacKay, 1993).

Early observation of dynamic changes on the levels of both Vtgs and vtg mRNAs in the
male Xenopus liver after estrogen treatment indicated that the expression of vtgs is
estrogen-dependent (Wallace and Jared, 1968; Baker and Shapiro, 1977). Furthermore, it

was first observed in male Xenopus that a second administration of estrogen caused a
quicker response, resulting in a higher level of Vtgs than that after the first administration
of estrogen (a so called “memory effect”) (Tata, 1988). The “memory effect” of estrogen

treatment was also observed in rainbow trout and tilapia (Le Guellec et al., 1988; Lim et
al., 1991). Lazier and MacKay (1993) proposed that after the primary estrogen exposure,
long-term alterations in either chromatin conformation or in transcription factors may be
responsible for the “memory effect”. Interestingly, temperature also modulates the
responsiveness of the teleost fish liver to estrogen treatment, with enhanced translational
or post-translational capacities at higher temperature (Lazier and MacKay, 1993).

A parallel increase in estrogen receptor (ER) was observed in male Xenopus liver after
estrogen treatment and the increased ER was equivalent to that in the female liver and
persisted for several weeks (Hayward et al., 1980). Studies using primary cultured
rainbow trout hepatocytes clearly showed that after estrogen treatment, a rapid increase

13
Chapter 1. General Introduction
(up to 15 fold) in the levels of both ER and its mRNAs was detected followed by
induction of Vtg and its mRNAs (Flouriot et al., 1996). Moreover, it was demonstrated
that the rainbow trout ER gene exhibits a low threshold response to loaded ER, which
contrasts with the expression of vtg. Vtg expression requires a higher loaded ER and its
transcriptional response is directly proportional to the amount of synthesized ER (Flouriot
et al., 1997).

Estrogen response element (ERE) has a consensus sequence of “GGTCANNNTGACC”,
which was found upstream of a chicken vtg and four Xenopus vtgs and demonstrated to
confer estrogen inducibility in vitro (Wahli, 1988 and reference within). Further studies
revealed that in the promoter region of Xenopus B1 gene, there are five binding sites for
the ubiquitous transcription factor CTF/NF-1 and multiple closely-spaced elements for

two liver-enriched factors, C/EBP and HNF3 (Cardinaux et al., 1994). These cis-acting
elements have been suggested to play important roles in regulating the estrogen-dependent
and liver-specific expression of Xenopus vtgs (Cardinaux et al., 1994; Kaling et al., 1991).
Similarly, three imperfect EREs and various binding sites for transcription factors GATA
and vitellogenin binding protein (VBP) have been identified in the promoter region of
tilapia vtg1 (Teo et al., 1998; Teo et al., 1999). Further studies indicated that both GATA
and VBP synergise with ER, which contributes to the hormone-dependent and tissue-
specific expression of the tilapia vtg (Teo et al., 1999).

Thus, liver-specific vtg expression in vertebrates is strictly regulated by estrogen at the
transcriptional level. At the post-transcriptional level, the estrogen-mediated stabilization
of vtg mRNAs also plays an important role in the regulation of the amount of vtg mRNAs

14
Chapter 1. General Introduction
in hepatocytes (Wahli, 1988). Dodson and Shapiro (1997) identified a vtg mRNA binding
protein, vigilin, which acts in the hormonal control of mRNA metabolism through binding
with the 3’-UTR of vtg mRNAs.

1.3. Gene transfer methods used in transgenic fish research
1.3.1. The importance of making transgenic fish
There are two main aspects of application of transgenic fish, aquaculture and basic
researches, such as developmental biology. Early studies of transgenic fish mainly
concentrated on improving beneficial traits of fish species for aquaculture. For example,
the transfer of growth hormone (GH) gene into various fish species has been carried out in
80-90s in order to produce “super fish” with enhanced growth rate (see review by Gong
and Hew, 1995). Also to enable cold-sensitive fish of commercial importance to be
farmed in cold region, transfer of antifreeze protein (AFP) gene has been carried out.
However, progress has been hampered by the low level of production of AFP in the
transgenic fish, though successful transgenic Atlantic salmon with the AFP gene have

been produced (Fletcher et al., 1988; Shears et al., 1991). Recently, concerns over
environmental issues and food safety have caused this area of research stagnate.

In sharp contrast to the situation encountered in applying transgenic fish to aquaculture,
the use of transgenic fish, especially popular model fish, the zebrafish (Danio rerio) and
medaka (Oryzias latipes), has gained increasing attentions in the basic research of
developmental biology in recent years and substantial progresses have been made (for
reviews, see Gong et al., 2001; Udvadia and Linney, 2003). The main reasons for this
success are the advantages of these model fish, having short generation time, rapid and

15
Chapter 1. General Introduction
externally developing transparent embryos compared with rodent models and the
opportunity of introducing of fluorescent report genes, such as green fluorescent protein
(GFP) gene in the fish. The application of GFP transgenic zebrafish in developmental
studies includes recapitulation of gene expression patterns and tissue/organ development,
analysis of regulatory elements of gene promoters/enhancers, tracing cell lineage and cell
migration, analysis of upstream regulatory genes, mutagenesis screening and
characterization, etc. (for detailed descriptions, see Gong et al., 2001). About 70 stable
lines of transgenic zebrafish have been produced worldwide, such as those with transgene
expressions in nervous system, lymphoid cells, epithelia, pancreas, muscle, blood, germ
cells, vasculature (Udvadia and Linney, 2003 and references within). These transgenic
zebrafish lines provide a rich resource in targeted screens in developmental analyzes
(Udvadia and Linney, 2003).

Other potential applications include using transgenic fish for biomonitoring, as ornamental
fish or as bioreactor. Udvadia and Linney (2003) proposed three approaches for detecting
environmentally harmful chemicals including using reporter transgenic fish transferred
with certain reporter genes which are under the control of chemically inducible promoters.
Gong et al. (2003) reported the development of three stable lines of transgenic zebrafish

with strong muscle expressions of GFP, yellow fluorescent protein (YFP) and red
fluorescent protein (RFP), respectively. These transgenic fish display vivid fluorescent
colors and the expression levels of fluorescent proteins range from 3% to 17% of total
muscle proteins. Thus, the transgenic fish can be used as new varieties of ornamental fish
and as a potential bioreactor system for recombinant protein production (Gong et al.,
2003).

16
Chapter 1. General Introduction

In conclusion, both aquaculture and research in developmental biology and environmental
toxicology have or will benefit from transgenic fish technology. Thus, gene transfer, as the
first step in making transgenic fish, is obviously very important. The techniques of gene
transfer vary and have progressively improved.

1.3.2. Current gene transfer techniques used in making transgenic fish
A. Microinjection
This technique was originally used in making transgenic mice by microinjection of foreign
DNA into mouse embryos (Gordon et al., 1980). Since then, microinjection has become
the most popular and efficient method for generation of transgenic fish, including the
zebrafish (Stuart et al., 1988), tilapia (Brem et al., 1988), rainbow trout (Guyomard et al.,
1989) and medaka (Matsumoto et al., 1992). After microinjection of plasmid DNA into
fertilized eggs, high molecular weight concatemers are immediately formed, which remain
in extrachromosomal state and are amplified by ~10-fold prior to gastrulation. During
gastrulation, the majority of foreign DNA is degraded and germline transformation
observed in 5-25% of injected fish (reviewed by Udvadia and Linney, 2003). Several
components are needed for setting up this gene delivery system, including a stereo-
microscope, a glass needle puller, a micromanipulator and an injection apparatus. Eggs
suitable for injection are at the one cell stage until the 2 to 4 cell stage. Thus, the working
window for microinjection is relatively short. In some fish, such as medaka, the pace of

early division of fertilized eggs can be slowed by keeping them at 4°C temporarily,
enabling a longer working window.


17
Chapter 1. General Introduction
B. Electroporation
When embryos or sperm are subjected to a series of brief electrical pluses, their cell
membranes become permeable to the surrounding DNA molecules, which then enter into
the cytoplasm and are finally integrated into the host genome. Large number of embryos
can be treated simultaneously and only an electrical pulse generator is required. A direct
current (DC) with either a pulse of exponential decay or multiple rectangle pulses was
used in early studies (for review, see Sin 1997). However, embryos with intact chorions
may not be efficiently transferred with foreign DNA and the rate of germ-line
transmission was low (Maclean and Rahman, 1994). In addition, a high mortality rate (55-
95%) of electroporated embryos was also reported (Powers et al., 1992). A recent study
indicated that DC-shifted radio frequency pluses greatly decreased the mortality to 30%
and increased the rate of germ-line transmission (60% of the electroporated embryos
passing the transgene on to their offspring) (Hostetler et al., 2003). This approach seems
promising in the future.

C. Sperm-mediated gene transfer
The first attempt using sperm as vectors for introducing foreign DNA into eggs was made
in mice by Lavitrano et al. (1989). In 1992, Khoo et al. produced transgenic zebrafish by
fertilization with sperm that had been directly incubated with plasmid DNA. Alternatively,
electroporated sperm exposed in the solution of exogenous DNA can be used (Muller et
al., 1992; Sin et al., 1993). The DNA transfer rate in this case is not very high. Although
sperm-mediated gene transfer has been successfully used to generate transgenic fish, full
harness of this gene transfer method will rely on the improvements of its reproducibility
and the foreign gene integration rate.

18
Chapter 1. General Introduction
D. Particle bombardment
Gene transfer through particle bombardment was first reported in 1980s. By this method,
cells are bombarded with high-velocity tungsten or gold particles coated with DNA, which
can penetrate cellular barriers and deliver DNA into cells and nuclei (Klein et al., 1987).
To accelerate the microprojectiles, a gas-shock inducer is needed. This method is
especially useful for transforming cells that are resistant to traditional transfer methods
such as microinjection and electroporation. The advantage of this approach is that a large
number of samples (200 to 1400 embryos) can be treated simultaneously (Zelenin et al.,
1991). However, a low germ-line transmission rate of foreign genes was observed
(Couble, 1997).

E. Lipofection
Liposome-mediated gene transfer (lipofection) involves formation of liposomes and DNA
entrapment process. The prevalent lipofection system involves cationic lipids, which have
the inherent problem of short circulating life and non-specificity. Late, cationic liposomes
coated with polyethylene glycol (PEG) were developed, which have attractive
characteristics such as preventing self-aggregation and reducing non-specific interactions
(Hong et al., 1997; Meyer et al., 1998). Lipofection has been used in vitro to transform
fish embryos (Szelei et al., 1994) and a large number of dechorionated eggs (200 to 400)
could be treated at a time (Szelei et al., 1994).

19
Chapter 1. General Introduction

F. Other techniques
Other gene delivery techniques include cell-mediated gene transfer using primordial germ
cells (PGCs) isolated from the genital ridges of hatching embryos of rainbow trout

(Takeuchi et al., 2003) and nuclear transplantation (Wakamatsu et al., 2001).

1.3.3. Drawbacks of the current gene transfer techniques
There are several drawbacks associated with the current gene transfer methods which may
be inefficient for routine transgenic fish research and limit the broad application of the
transgenic technologies in additional fish species. First, experienced personnel are
required for some of the gene delivery techniques such as microinjection. Second, special
equipment is indispensable, i.e. for microinjection, electroporation and particle
bombardment. Third, not all methods are applicable for treatment of a large number of
fish eggs at a time. Forth, most gene delivery methods are limited by the structure of fish
eggs. The small nuclei of fish eggs are difficult to visualize, chorions hardened soon after
fertilization and eggs in many species of fish are totally totally opaque. Some fish eggs
may be too fragile and prone to be damaged by microinjection and other gene delivery
methods. Moreover, some fish species do not produce free eggs for in vitro manipulation
as embryonic development occurs in fish’s mouth. Thus, alternative gene delivery
approaches need to be developed. Ideally, the new gene delivery approach will be easy to
perform, does not depend on special equipment, is not laborious and is not limited by the
structure and reproduction behavior of fish eggs.

1.4. Receptor-mediated gene transfer

20
Chapter 1. General Introduction
In animal cells, one of the mechanisms that facilitates the internalization of specific
substances into cells is the process called receptor-mediated endocytosis (RME) which
provides a major pathway for trafficking of extracellular molecules (or ligands) into cells.
RME involves the binding of a ligand to a specific cell surface receptor, followed by
membrane invagination, formation of internal vesicle and delivery of vesicle to
cytoplasmic organelles, such as endosome (Karp, 1996). Many ligands and their receptors
involved in the RME system have been identified, such as the low density lipoprotein

(LDL) and its receptor, asialoglycoprotein (ASGP) and its receptor(for review, see
Schwartz, 1995).

Based on the RME process, a novel gene transfer approach called receptor-mediated gene
transfer has been proposed and verified by Wu and Wu (1987). Initially, they
demonstrated the feasibility of this gene delivery approach in vitro using cultured mouse
hepatocytes. Briefly, conjugates were constructed by coupling asialoorosomucoid (ASOR)
to poly-L-lysine (pLys) using N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP), and
complexes were prepared by mixing the conjugates with DNA. After incubating two types
of hepatocytes [asialoglycoprotein receptor (+) and (–) cells] with the complexes, they
demonstrated that the receptor (+) cells were transformed by the foreign DNA, while the
receptor (–) cells were not. In addition, neither cell type was transformed after incubation
with the mixtures of individual components of the complexes. Thus, they concluded that
the foreign DNA was transported into the hepatocytes by the asialoglycoprotein receptor-
mediated pathway (Wu and Wu, 1987). Subsequent progress has been made by this group
such as successfully applying this receptor-mediated gene delivery approach to living
animals (Wu and Wu, 1988), detection of persistent expression of a delivered foreign gene

21
Chapter 1. General Introduction
in the rat liver (Wu et al., 1989) and partial correction of a genetic defect in rat using this
gene delivery technology (Wu et al., 1991). Gene delivery based on similar approaches
was also reported by other groups. For example, transferrin has been covalently linked to
protamine or polylysine to form DNA binding conjugates, which can mediated DNA
uptake by avian erythroblast cells (Wagner et al., 1990). Chen et al. (1994b) reported that
a monoclonal antibody to the human epidermal growth factor (EGF) receptor was
conjugated with polylysine and the resulting conjugates could deliver DNA into EGF
receptor-overproducing squamous carcinoma cells by receptor-mediated endocytosis.

Receptor-mediated gene transfer has been extensively studied in mammalian systems and

is a promising gene delivery technique in human gene therapy (for review, see Swaan,
1998; Zauner et al., 1998; Uherek and Wels, 2000). There are several advantages of this
gene delivery system, such as specific cell type targeted, utilization of non-viral vectors
and suitability for in vivo delivery, in spite of its transient nature of transfection and
relatively low efficiency (Zauner et al., 1998).


1.5. Rationale and objectives of the current study
1.5.1. Rationale of the study
Although receptor-mediated gene transfer has been extensively used in mammalian
systems and a wide range of ligands have been tested for the purpose of delivering genes
to different types of cells (Zauner et al., 1998), there has been no report regarding
utilization of this gene transfer approach in producing transgenic fish.


22
Chapter 1. General Introduction
In teleost fish, it is well known that the major egg yolk protein precursor vitellogenin is
synthesized in the liver under the control of E
2
and incorporated into oocytes via receptor-
mediated endocytosis (Selman and Wallace, 1982; Tyler et al., 1987; Tyler et al.,
1988a&b; Chan et al. 1991). Thus, Vtg is an ideal candidate protein as DNA delivery
vehicles if the fish oocytes are targeted in gene transfer in vivo through the receptor-
mediated gene delivery approach, as previously suggested by Gong and Hew (1995). If
this gene delivery approach is feasible in fish, foreign genes can be specifically introduced
into the oocytes through receptor-mediated endocytosis of the Vtg-DNA complexes after a
simple injection of the complexes into fish blood circulation. This gene delivery approach
is easy and time-saving compared with other approaches, in that one injection into the
founder female fish may result in thousands of transgenic offspring (depending on the

number of eggs the fish can produce), which will undoubtedly expedite the establishment
of stable transgenic lines. If traditional plasmid DNAs used in this gene delivery system
are to be replaced by pseudotyped retroviral vectors or transposon based constructs, a
higher rate of germ-line transmission of introduced foreign genes may be expected, since
these two types of vectors have been proven to render a high frequency of chromosome
integration (Gaiano et al., 1996; Izsvak and Lvics, 2004)

The proposed receptor-mediated gene transfer used in a teleost fish system involves
utilization of teleost Vtgs as DNA carrier. Based on the available sequence information, it
seems that teleost vtg genes also belong to a multigene family containing divergent vtg
members. Thus, whether purified native Vtgs or recombinant Vtgs are to be used in
preparation of DNA carrier, it is appropriate to characterize the vtg multigene family in the

23
Chapter 1. General Introduction
experimental fish first and determine which vtg gene and its translation products are
proper candidates for the subsequent receptor-mediated gene transfer study.

1.5.2. Experimental fish used in this study
Two species of teleost fish, the zebrafish (Danio rerio) and the red tilapia (Oreochromis
mossambica), were used to characterize the vtg multigene family and develop a receptor-
mediated gene transfer method, respectively, in the present study.

The zebrafish is a small freshwater fish, which is easily available from pet shops. The cost
for maintaining this fish is cheaper than most other laboratory animals. The zebrafish has a
relative short generation time of about 3 months and can lay a large number of eggs (100-
200) per mating. Completely external development of its embryos provides easy access to
all developmental stages. Most importantly, zebrafish embryos are optically transparent
through the early developmental stages. Therefore, embryo manipulation and screening
become easier in zebrafish than in most other experimental models. The zebrafish has

attracted the attention of many researchers and has been intensively used in developmental
and genetic studies for over two decades resulting in a more detailed genetic background
for this fish than any other fish species. Thus, the zebrafish is an ideal model for
characterizing reproduction related genes such as vtg genes.

Furthermore, a cDNA library from adult zebrafish has been constructed in our lab (Gong
et al., 1997), which provides a rich source for identification of zebrafish vtgs by the
expressed sequence tag (EST) approach. A radiation hybrid panel T51 is commercially
available from Research Genetics and provides a powerful tool for genome mapping in

24
Chapter 1. General Introduction
zebrafish. In addition, abundant sequence information of zebrafish ESTs and genes is
available publicly (zebrafish EST database at UniGene database
facilitating sequence
comparison and gene copy number determination. Most importantly, Ensembl zebrafish
genome database v19.3.2 ( has been released.
Although it is still not complete, the zebrafish genome database contains valuable
information about gene organization and chromosome locations, which facilitates
identification of any potential novel zebrafish vtgs and the determination of linkage
relationships between different vtgs.

However, due to the small size, it is not feasible to inject vitellogenin-DNA complexes
into blood vessel of the zebrafish. Thus, the trial of receptor-mediated gene transfer was
carried out in the red tilapia (Oreochromis mossambica), as the tilapia fish is relatively
large in size. Thus, it is much easier to perform injection through the caudal artery in the
tilapia than in the zebrafish.


1.5.3. Objectives of the study

There are two main objectives in this study:
1) Characterization of vtg multigene family in the zebrafish. Topics related to vtgs will be
investigated including gene copy number, nucleotide and deduced amino acid sequences,
chromosome locations, evolutionary relationships between teleost vtgs, the expression
sites and E
2
inducibility of vtgs. From this study, candidate vtg and its protein will be
chosen for the development of receptor-mediated gene transfer in teleost fish.

25