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a Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato-ku, Tokyo 108-8477, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, developmental biology, early development, gametogenesis, gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell-mediated gene transfer could be an important tool for producing transgenic fish, especially in commercially valuable species that have a large body size and a long generation time. The conventional gene transfer techniques in fish usually result in the random integration of transgenes, which causes a high degree of mosaicism and an unpredictable expression pattern of the transgene [4]. Thus, rigorous screening of possible founder fish and F1 fish, which is a laborious and expensive process, is needed to establish transgenic strains with desirable traits. However, with a cell-mediated gene transfer system, it would be possible to deliver a foreign gene into the genome of cultured cells and to isolate only the transformants by drug selection before they are converted into individual fish. This approach would avoid the many drawbacks of conventional methods and would provide a reproducible and reliable method for producing genetically modified fish in commercially valuable species. Moreover, this system would make it possible to produce gene-targeted fish via homologous recombination.
To establish PGC-mediated gene transfer in commercially important fish such as salmonids, a technique for mass isolation of PGCs is necessary. However, convenient PGC markers, such as alkaline phosphatase activity or antibodies against PGC-specific cell surface antigens that are available in mammals and birds, are not available in fish [5]. Thus, no methods for isolating PGCs in fish are available. Recently, the germline marker vasa was characterized in zebrafish [6, 7]. Also a vasa-like homologue was found in rainbow trout (Oncorhynchus mykiss). This homologue, rainbow trout vasa-like gene (RtVLG), was cloned and was shown to be expressed only in the germ cell lineage [8]. We have characterized RtVLG regulatory regions using a green fluorescent protein (GFP) gene as a reporter. The construct (pvasa-GFP), which contains the 5' and 3' flanking sequences and the first intron of the RtVLG gene, involved all essential cis-elements required for PGC-specific gene expression [9].
In this study, for the mass production of transgenic embryos that express stable green fluorescence in their germ cells, we established transgenic trout strains carrying pvasa-GFP. To obtain live PGCs as material for in vitro cell culture, we developed a technique to purify a large number of viable fish PGCs by fluorescence-activated cell sorting.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The structure of the transgene (pvasa-GFP) and the microinjection procedure were previously reported [9]. The pvasa-GFP transgene was constructed using the 4.7-kilobase (kb) 5' fragment of RtVLG, an enhanced GFP gene (Clontech, Palo Alto, CA), the 3' untranslated region derived from RtVLG cDNA, and a 1.5-kb 3' flanking region. The 5' fragment contained 3.3 kb of the 5' flanking region, 130 base pairs (bp) of the first intron (which contains only the 5' untranslated sequences), 1.3 kb of the first intron, and a part of the second exon including the start codon. Semen were collected from 1-yr-old transgenic founders. DNA was extracted from 2 nl of semen using a GenomicPrep Cells and Tissue DNA Isolation Kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's protocol. The sperm DNA from each individual was subjected to polymerase chain reaction (PCR) with GFP-specific primers, according to the method of Takeuchi et al. [10]. Founder males producing PCR-positive semen were used for the following studies.
Production of Stable Transgenic Lines and Analysis of GFP Expression
To generate F1 offspring, the eggs from wild-type females were inseminated with sperm from the PCR-positive founder males. In each founder fish, approximately 300 hatching embryos (35 days postfertilization ["f]) were screened for GFP expression under a fluorescence dissecting microscope (SZX-RFL; Olympus, Tokyo, Japan) equipped with a GFP filter set (DM505; Olympus). Transgenic F1 fish, which showed green fluorescence in their PGCs, were collected and raised to sexual maturity. Semen from an F1 transgenic male was used to fertilize wild-type eggs to produce F2 offspring. The precise localization and the morphology of GFP-expressing cells were investigated by immunohistochemistry employing an antibody specific to GFP as previously reported [9]. Whole-mount in situ hybridization with an RtVLG antisense probe was performed using the method of Yoshizaki et al. [8].
Dissociation of Genital Ridges and Fluorescence-Activated Cell Sorting
Under the dissecting fluorescence microscope, genital ridges from hatching embryos were manually dissected using watchmaker's forceps. The collected genital ridges were incubated with trypsin (Worthington Biochemical Corp., Lakewood, NJ; 0.5% in PBS, pH 8.2) for 2 h at 20°C. The resulting cell suspension was filtered through a nylon mesh with 42-µm pore size. GFP-positive and -negative cells were sorted by flow cytometry using an EPICS Elite Cell Sorter (Beckman-Coulter, Miami, FL). Sorted cells were collected in Dulbecco modified Eagle medium (Life Technologies, Rockville, MD) with 10% fetal calf serum. Determination of purity among the sorted cells was based on counting at least 100 cells per experiment to estimate the percentage of fluorescent cells. To examine RtVLG expression in the sorted cells, total RNA was extracted from the sorted cells using Isogen (Nippon Gene, Tokyo, Japan). Reverse transcription (RT) reactions were performed using Ready-To-Go You-Prime First-Strand Beads (Amersham Pharmacia) according to the manufacturer's protocol. RT-PCR was performed on the resulting cDNA products with RtVLG-specific primers [8]. The PCR amplification was conducted in 10 µl of reaction mixture, containing 0.5 µl of cDNA products, 200 µM of dNTPs, 10 pmol each of primers, and 0.25 units of ExTaq polymerase (Takara Biomedicals, Osaka, Japan) in the supplied buffer. The PCR was carried out at 94°C for 30 sec, 64°C for 30 sec, and 94°C for 20 sec for 40 cycles with a 3-min initial 94°C denaturation step and a 3-min final elongation step. ß-Actin was used as an internal control for RT-PCR with rainbow trout ß-actin cDNA specific primers (5'-ACT ACC TCA TGA AGA TCC TG-3' and 5'-TTG CTG ATC CAC ATC TGC TG-3').
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GFP expression in putative PGCs was observed in microinjected (P1) embryos at the eyed stage, at 25 dpf (data not shown). In all P1 embryos examined, GFP expression in PGCs was highly mosaic. Although some of the P1 embryos expressed GFP only in PGCs, relatively large numbers of embryos showed ectopic GFP expression besides its expression in PGCs. Therefore, we used nonmosaic F1 or F2 transgenic embryos for further studies. To do so, the P1 generation was raised to sexual maturity. Semen of 1-yr-old male trout was collected and analyzed by PCR with GFP-specific primers. Sperm DNA samples were obtained from 117 P1 males. Of these samples, 30 showed GFP-specific signals. Three hundred F1 hatching embryos derived from each of the 30 PCR-positive males were examined under the fluorescence microscope. Embryos in 9 of the 30 lines harbored clusters of GFP-expressing cells located above the digestive tract (Fig. 1A). In each F1 sibling derived from the nine transgenic founders, the appearance rates of GFP-expressing embryos ranged from 1% to 8%. An immunohistochemical analysis with GFP-specific antibody revealed that GFP-expressing cells were relatively larger than the other cells and were located in the genital ridges (Fig. 1C).
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GFP Expression in F2 Developing Embryos
F2 embryos were produced by mating a transgenic F1 male with a nontransgenic female. GFP expression in PGCs was observed in approximately 50% of these embryos. At the 40-somite stage (10 dpf), GFP fluorescence was barely detectable in putative PGCs (data not shown). GFP fluorescence of PGCs became more intense at the 70-somite stage (14 dpf) (Fig. 2A). At this stage, GFP-expressing cells were located on the midline and formed a single cluster just above the intestine. By the eyed stage, GFP-expressing cells had migrated anteriorly and appeared in two bilateral rows (Fig. 2C). The number of GFP-expressing cells did not appear to increase during this migration period. In the 70-somite and eyed stages, the localization patterns and the numbers of the GFP-expressing cells coincided with those of RtVLG mRNA (Fig. 2, B and D). Strong green fluorescence still appeared in germ cells of immature testis (Fig. 2E) and ovary (Fig. 2F) of 8-mo-old fish.
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Purification of GFP-Labeled Primordial Germ Cells by Fluorescence-Activated Cell Sorting
Because the GFP gene was specifically expressed in PGCs (Fig. 3A), fluorescence-activated cell sorting was performed to isolate a pure population of PGCs for in vitro studies. Manually excised genital ridges (Fig. 3B) from hatching embryos were dispersed by trypsin treatment. Cell suspensions consisted of GFP-positive PGCs and GFP-negative somatic cells (Fig. 3C). In the flow cytometric analysis, the GFP-positive and GFP-negative cell populations were clearly distinguished by means of their fluorescence intensity (Fig. 3D). Approximately 0.5% of the total input cells were GFP positive. Furthermore, we collected the GFP-positive cell population on the basis of its high fluorescence intensity. The average viability of dissociated cells after the trypsin treatment, estimated by trypan blue staining, was 90.3%. The percentages of GFP-positive cells in the sorted live cells were greater than 99% in three independent trials, as determined by fluorescence microscopy. We obtained 500 GFP-expressing cells from 20 embryos in the three trials. The GFP-positive cells were large (approximately 20 µm in diameter) and round, with eccentrically placed nuclei and a granule-rich cytoplasm. The extracted RNAs from the GFP-positive and GFP-negative populations were subjected to RT-PCR with RtVLG-specific primers. The predicted bands of 219 bp were detected in the GFP-positive cell populations but not in the GFP-negative cell populations (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GFP-labeled PGCs have also been isolated from transgenic mice carrying a GFP gene driven by the regulatory regions of the Oct-4 gene (Oct-4/GFP) [13], which is required for the undifferentiated state of embryonic cells. If the cells isolated from Oct-4/GFP mice were used for an in vitro culture study, it would be difficult to distinguish between cells possessing characters of the germ cell lineage and pluripotent cells dedifferentiated from germ cells, such as embryonic germ (EG) cells, by the presence or absence of green fluorescence. However, with the transgenic trout strains established in this study, we could easily distinguish between cells that had maintained a germline status and dedifferentiated cells because the expression of GFP was controlled by the regulatory elements of RtVLG, which is activated only in the germ cell lineage. The use of GFP as a real-time, noninvasive indicator of the status of a germ cell lineage is particularly convenient for establishing a cell line that retains its germline competency in organisms, including fish, whose ES cells and EG cells do not have the potency to differentiate into a germline. Ma et al. [2] reported that vasa expression is a good indicator of germline competency of zebrafish ES-like cells. Therefore, the GFP-labeled PGCs isolated in this study can be used to optimize in vitro culture conditions for establishing a cell line that can give rise to functional gametes.
In this study, rainbow trout was used as a model species because of its high fecundity and large embryo size. Fish PGCs do not proliferate during migration to the genital ridges [12], as seen in mice [14], and are present in very small numbers when they settle in genital ridges. At this developmental stage, the number of PGCs per embryo is about 50 in zebrafish [15], about 40 in medaka [16], and about 40–60 in rainbow trout [8]. Therefore, for mass isolation of fish PGCs, ordinary model fish species such as zebrafish and medaka are not as useful as fish species whose fecundity is high, such as salmonids. We could easily produce >1500 transgenic embryos harboring GFP-labeled PGCs by a single insemination using an F1 heterologous transgenic fish. For effective cell sorting with flow cytometry, a high concentration of target cells in the initial cell suspension is preferable. However, the number of PGCs in fish is very small, which leads to the necessity of using isolated genital ridges containing PGCs rather than whole embryos. Taking advantage of the large embryos of rainbow trout (15 mm in total length at hatching), it was not difficult to dissect hatching embryos to excise genital ridges using green fluorescence as an indicator. The collected genital ridges made it possible to prepare a PGC-rich cell population. Consequently, GFP-labeled PGCs were effectively purified by flow cytometry in a relatively short time. Thus, rainbow trout is an ideal model for mass isolation of fish PGCs.
In the present study, we routinely obtained 500 PGCs from 20 hatching embryos. Because the average (±SD) number of GFP-labeled PGCs at hatching (19 embryos) was 42 ± 12 (unpublished data), the predicted number of PGCs isolated from 20 embryos is about 840. Therefore, the recovery efficiency of PGCs by flow cytometry was about 60%. The purity and viability of isolated PGCs and the reproducibility obtained in this study were as high as those in mice [17, 18]. The present system for mass isolation of PGCs developed in this study should provide new opportunities to conduct cellular and molecular studies of fish germline stem cells. Abe et al. [19] reported that as few as 200 mouse PGCs were sufficient to construct a cDNA library using a PCR strategy. Complementary DNA libraries generated from various stages of germline development should provide valuable molecular markers of the fish germ cell lineage, which is particularly important for monitoring the differentiation of cultured PGCs in vitro.
In this study, we conducted the first mass purification of viable PGCs in ectothermal vertebrates. To use fish PGCs for cell-mediated gene transfer, future studies with respect to long-term cultivation and transplantation of PGCs are needed. We are presently attempting to develop techniques to cultivate PGCs in such a way that GFP fluorescence is maintained. We are also presently using the cell transplantation procedure developed for rainbow trout embryos [20] to transplant isolated PGCs for producing germline chimeras of rainbow trout. The GFP-labeled PGCs should help to trace the behavior of PGCs, including their distribution, proliferation, and differentiation, in recipient embryos.
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FOOTNOTES |
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Received: 18 March 2002.
First decision: 3 April 2002.
Accepted: 26 April 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  A. Yano, K. Suzuki, and G. Yoshizaki Flow-Cytometric Isolation of Testicular Germ Cells from Rainbow Trout (Oncorhynchus mykiss) Carrying the Green Fluorescent Protein Gene Driven by Trout vasa Regulatory Regions Biol Reprod, January 1, 2008; 78(1): 151 - 158. [Abstract] [Full Text] [PDF]
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G. Yoshizaki, Y. Tago, Y. Takeuchi, E. Sawatari, T. Kobayashi, and T. Takeuchi Green Fluorescent Protein Labeling of Primordial Germ Cells Using a Nontransgenic Method and Its Application for Germ Cell Transplantation in Salmonidae Biol Reprod, July 1, 2005; 73(1): 88 - 93. [Abstract] [Full Text] [PDF]
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Y. Takeuchi, G. Yoshizaki, and T. Takeuchi Generation of Live Fry from Intraperitoneally Transplanted Primordial Germ Cells in Rainbow Trout Biol Reprod, October 1, 2003; 69(4): 1142 - 1149. [Abstract] [Full Text] [PDF]
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