Chapter General Introduction Chapter General Introduction Chapter 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 receptormediated 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) Chapter 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 (E2) 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 Chapter 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 Chapter 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, hepaticallyderived Vtgs are the principle precursors of yolk proteins and their synthesis in the liver is in response to the circulating E2 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) Chapter 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) Chapter 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, E2 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 Chapter General Introduction vertebrates studied so far are in the form of dimers with molecular weights between 326550 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 Chapter 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 (apoLpII/I), human apolipoprotein B (apoB) and the large subunit of mammalian microsomal Chapter 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 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 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 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 tissuespecific 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 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 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 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 stereomicroscope, a glass needle puller, a micromanipulator and an injection apparatus Eggs suitable for injection are at the one cell stage until the to 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 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 (5595%) 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 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 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 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 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 receptormediated 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 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 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 E2 and incorporated into oocytes via receptormediated 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 receptormediated 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 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 receptormediated 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 months and can lay a large number of eggs (100200) 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 General Introduction zebrafish In addition, abundant sequence information of zebrafish ESTs and genes is available publicly (zebrafish EST database at http://zfish.wustl.edu/; UniGene database http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene), facilitating sequence comparison and gene copy number determination Most importantly, Ensembl zebrafish genome database v19.3.2 (http://www.ensembl.org/Danio_rerio/) 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 E2 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 Chapter General Introduction 2) Investigation of the potential application of Vtg system in gene transfer by the receptormediated approach This will include construction of DNA carrier, testing its efficiency in mediating DNA delivery by receptor-mediated approach and identification of receptorbinding domain in teleost Vtg 26 ...Chapter 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. .. harness of this gene transfer method will rely on the improvements of its reproducibility and the foreign gene integration rate 18 Chapter General Introduction D Particle bombardment Gene transfer. .. levels in the liver of teleost fish (Mommsen and Walsh, 19 88) 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