Lianchun Fan

Production of zebrafish germ-line chimeras from embryo cell cultures

Although the zebrafish possesses many characteristics that make it a valuable model for genetic studies of vertebrate development, one deficiency of this model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem cell cultures are routinely used for gene transfer and provide the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. Transgenic animals possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of a chimeric embryo. Although zebrafish embryo cell cultures that exhibit characteristics of embryonic stem cells have been described, successful contribution of the cells to the germ-cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures maintained in the presence of cells from a rainbow trout spleen cell line (RTS34st) are able to produce germ-line chimeras when introduced into a host embryo. Messenger RNA encoding the primordial germ-cell marker, vasa, was present for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cell differentiation in the embryo cell cultures. These results suggest that the RTS34st splenic–stromal cell line will be a valuable tool in the development of a cell-based gene transfer approach to targeted gene inactivation in zebrafish.

Homologous recombination in zebrafish ES cells

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.

Zebrafish Primordial Germ Cell Cultures Derived from vasa::RFP Transgenic Embryos

Although embryonic germ (EG) cell-mediated gene transfer has been successful in the mouse for more than a decade, this approach is limited in other species due to the difficulty of isolating the small numbers of progenitors of germ cell lineage (PGCs) from early-stage embryos and the lack of information on the in vitro culture requirements of the cells. In this study, methods were established for the culture of PGCs obtained from zebrafish embryos. Transgenic embryos that express the red fluorescent protein (RFP) under the control of the PGC-specific vasa promoter were used, making it possible to isolate pure populations of PGCs by fluorescence-activated cell sorting (FACS) and to optimize the culture conditions by counting the number of fluorescent PGC colonies produced in different media. Cultures initiated from 26-somite-stage embryos contained the highest percentage of PGCs that proliferated in vitro to generate colonies. The effect of growth factors, including Kit ligand a and b (Kitlga and Kitlgb) and stromal cell-derived factor 1a and 1b (Sdf-1a and Sdf-1b), on PGC proliferation was studied. Optimal in vitro growth and survival of the zebrafish PGCs was achieved when recombinant Kitlga and Sdf-1b were added to the culture medium through transfected feeder cells, resulting in a doubling of the number of PGC colonies. Results from RT-PCR and in situ hybridization analysis demonstrated that PGCs maintained in culture expressed the kita receptor, even though receptor expression was not detected in PGCs isolated by FACS directly from dissociated embryos. In optimal growth conditions, the PGCs continued to proliferate for at least 4 months in culture. The capacity to establish long-term PGC cultures from zebrafish will make it possible to conduct in vitro studies of germ cell differentiation and EG cell pluripotency in this model species and may be valuable for the development of a cell-mediated gene transfer approach.

Zebrafish embryonic stem cells.

Methods are presented for the derivation of zebrafish embryonic stem (ES) cell cultures that are initiated from blastula and gastrula stage embryos. To maintain pluripotency, the ES cells are cocultured with rainbow trout spleen cells from the RTS34st cell line. ES cells maintained for multiple passages on a feeder layer of growth-arrested RTS34st exhibit in vitro characteristics of pluripotency and produce viable germ cells following transplantation into a host embryo. The ES cells are able to undergo targeted plasmid insertion by homologous recombination, and methods are described for the introduction of a targeting vector by electroporation. Two strategies are described for the efficient isolation of homologous recombinants using a visual marker screen and positive-negative selection.

Zebrafish embryo cells remain pluripotent and germ-line competent for multiple passages in culture.

Mouse embryonic stem (ES) cell lines are routinely used to introduce targeted mutations into the genome, providing an efficient method to study gene function. Application of similar gene knockout techniques to other organisms has been unsuccessful due to the lack of germ-line competent ES cell lines from non-murine species. Previously, we reported the production of zebrafish germ-line chimeras using short-term primary embryo cell cultures. Here we demonstrate that zebrafish embryo cells, maintained for several weeks and multiple passages in culture, remain pluripotent and germ-line competent. Zebrafish germ-line chimeras were generated from passage 5 and 6 cultures initiated from blastula- and gastrula-stage embryos. In addition to the germ line, the cultured cells contributed to multiple tissues of the host embryo, including muscle, liver, gut, and fin. To facilitate the identification of germ-line chimeras, ES cells expressing the green fluorescent protein (GFP) were introduced into host embryos, and germ-line contribution was detected by the presence of GFP+ cells in the region of the gonad. The germ-line competent embryo cell cultures will be useful for the development of a gene targeting strategy that will increase the utility of the zebrafish model for studies of gene function.

Initiation of a Zebrafish Blastula Cell Line on Rainbow Trout Stromal Cells and Subsequent Development Under Feeder-Free Conditions into a Cell Line, ZEB2J

A continuous cell line, ZEB2, was developed from zebrafish blastula-stage embryos expressing enhanced green fluorescent protein (GFP). Originally the rainbow trout spleen cell line, RTS34st, was used as feeders to initiate and maintain the cells through several passages. ZEB2 was then grown for 2 years without feeders in L-15 with 15% fetal bovine serum (FBS) for 120 population doublings. This new cell line, ZEB2J, was heteroploid, had detectable telomerase activity, and was adherent. After growing into monolayers, some cells continued to grow into mounds. Cultures expressed Pou-2 mRNA and contained many alkaline phosphatase and a few stage-specific embryonic antigen-1-positive cells. In dishes coated with a phospholipid polymer (2-methacryloxyloxyethyl phosphorylcholine, MPC), ZEB2J formed spherical aggregates. Aggregates attached to conventional culture plastic, and most cells that emerged from aggregates had typical epithelial-like shapes of ZEB2J, which suggests that ZEB2J had limited differentiation potential, despite expressing some stem cell properties. The fluorescence of ZEB2J allowed relationships with feeder cells to be studied. In MPC dishes, ZEB2J formed mixed spheroids with RTS34st. In adherent cocultures, RTS34st and other fish cell lines strongly stimulated the ZEB2J growth, which could be quantified specifically because ZEB2J expressed GFP. ZEB2J should be useful for optimizing culture conditions for zebrafish embryonic stem cells.

Culture of Embryonic Stem Cell Lines from Zebrafish

Publisher Summary This chapter describes the culture of Embryoniv stem cell lines from Zebrafish. Despite its many advantages for studies of embryo development and human disease, one deficiency of the zebrafish model has been the lack of methods for targeted mutagenesis using embryonic stem (ES) cells. A similar strategy applied to zebrafish would complement other genetic methods currently available, such as large-scale random mutagenesis antisensebased gene knockdown and target selected mutagenesis approaches to increase the utility of this model system. To address this problem, our laboratory has been working to establish zebrafish ES cell lines that are suitable for use in a gene targeting approach. This chapter illustrates that the germline competent ES cells are genetically altered in culture by targeted incorporation of foreign DNA by homologous recombination followed by in vitro selection of cell colonies that have undergone the targeting event. This chapter describes the methods for the derivation of germline competent zebrafish ES cell cultures along with a protocol for the efficient introduction of plasmid DNA into the cells by electroporation and in vitro selection of homologous recombinants.

Production of Zebrafish Germline Chimeras by Using Cultured Embryonic Stem (ES) Cells

Publisher Summary This chapter focuses on the production of zebrafish germline chimeras by using cultured embryonic stem (ES) cells. The development of an embryonic stem (ES)-cell-mediated approach to gene targeting in zebrafish require that methods be available for the production of germline chimeras. To generate a knockout, ES cells carrying the targeted mutation are selected in vitro and introduced into recipient embryos to produce germline chimeras, which are then bred to establish the mutant line. The capacity of ES cells to contribute to the germ cell lineage of a host embryo is maintained when the ES cells are cultured on a feeder layer of growth-arrested rainbow trout spleen cells. In addition to the germline, ES cells contribute to multiple tissues of the chimeric host. Although the frequency of germline chimera production is low (approximately 2–4%), the ability to conveniently inject a large number of embryos with ES cells makes it feasible to produce a sufficient number of germline chimeras to establish a transgenic or knockout line of fish. To facilitate the identification of germline chimeras, a zebrafish ES cell line is established from transgenic embryos that constitutively express the green fluorescent protein. Several strategies are currently being pursued to improve the efficiency of germline chimera production from zebrafish ES cell cultures.

47 – Isolation and Culture of Zebra Fish ES Cells

This chapter describes methods for derivation, maintenance, and genetic manipulation of zebra fish embryonic stem (ES) cell cultures. A key component of the cell culture system is the use of feeder layers, derived from rainbow trout spleen cell lines. Methods are described for the introduction of a targeting vector by electro oration with procedures for the efficient selection of homologous recombinants using a visual marker screen. Also, a protocol is presented for the introduction of the ES cells into host embryos by microinjection to generate zebra fish germ-line chimeras. Reverse genetic methods have also been used to study zebra fish gene function. Colonies of ES cells that harbor targeted mutation are selected and expanded in culture and the cells are introduced into a host embryo, where they participate in development and contribute to the germ cell lineage. Development of an ES cell-based gene targeting system in zebra fish requires the cells to be able to incorporate vector DNA in a targeted fashion by homologous recombination and those methods are available to identify and select the colonies of homologous recombinants.

Use of Cultured Primordial Germ Cells for Production of Transgenic Fish

Due to its favorable characteristics, the zebrafish is a popular model of vertebrate development. However, one deficiency of the zebrafish model system is the lack of methods for cell-mediated gene transfer and targeted mutagenesis. In mice, cell-mediated gene transfer is accomplished through the use of embryonic stem (ES) cell cultures and provides the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. ES cells possessing a targeted mutation are selected in culture and transferred to a host embryo. Transgenic mice possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of the chimeric embryo. In zebrafish, embryo cell cultures have been derived that exhibit in vitro characteristics of ES cells but successful contribution of the cells to the germ cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures, maintained in the presence of cells from a rainbow trout spleen cell line, RTS34st, are able to produce germ line chimeras when introduced into a host embryo. Zebrafish embryo cells co-cultured with RTS34st cells or their conditioned medium continue to possess mRNA encoding the primordial germ cell marker, vasa, for more than 30 days. In the absence of RTS34st cells or conditioned medium the vasa mRNA disappeared by five days in culture. The spleen cells also inhibited the embryo cell cultures from differentiating into melanocytes and neuronal cell types. The influence of the RTS34st cells on the zebrafish embryo cell cultures indicate that the splenic stromal cell line will be a valuable tool in the application of cell-mediated gene transfer and targeted gene inactivation technology to zebrafish.