Development of a positive–negative selection procedure for gene targeting in fish cells

Chen, Songlin; Hong, Yunhan; Schartl, Manfred

doi:10.1016/s0044-8486(01)00811-0

Cited by 19 publications

(27 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The key to the PNS is the negative selection, and the best selectable marker in mammalian cells is the HSVtk gene. Recently, we have shown that the PNS using neo and HSV-tk is effective in fish cells, resulting in an enrichment of 2-and 60-fold in the melanoma cell line PSM and the epithelioma papulosum cyprini cell line EPC (Chen et al, 2002). In this study, numerous vectors have been constructed and selection conditions have been developed.…”

Section: Discussionmentioning

confidence: 99%

“…Cui et al (2003) have reported RecA-mediated, targeted mutagenesis in zebrafish. Previously we have shown the possibility of drug selection for gene transfer into established piscine non-ES cell lines (Chen et al, 2002). Here we report the optimization of conditions for efficient gene transfer, reporter assay and drug selection in the medaka ES cell line MES1 that has the potential to be used for gene targeting in fish.…”

Section: Introductionmentioning

confidence: 93%

“…Plasmid pHygEGFP expresses a fusion for hygromycin resistance and EGFP (Clontech), and pCMVpac:tk expresses a fusion for puromycin acetyltransferase (PAC) and HSV-TK and has been tested in mammalian cells (Karreman, 1998). Cassettes pSTneo, pSTk and pNK express neo, tk and neo plus tk respectively from the regulatory sequence ST, consisting of one SV40 early gene enhancer linked to the TK promoter (Chen et al, 2002). Briefly, the neo gene together with two polyadenylation signals was cut off Figure 1.…”

Section: Selectable Gene Cassettesmentioning

confidence: 99%

“…One day later, the cells were exposed to drugs G418 (400 µg/ml), hygromycin (400 µg/ml), puromycin (1 µg/ml), Gc (2 µM), puromycin plus Gc (for cells transfected by pCMVpac:tk) or G418 plus Gc (for cells transfected by pNK). After 18-25 days of continuous culture with regular medium changes, colonies were either picked up manually and transferred to multiwell plates for expansion or fixed in methanol-acetic acid (3:1) for counting after staining with 5% Giemsa (Chen et al, 2002).…”

Section: Drug Selectionmentioning

confidence: 99%

“…Recently, neo and HSVtk have been tested in two piscine non-ES cell lines (Chen et al, 2002). Similarly, when MES1 cells were seeded at 2 × 10 7 in 10-cm dishes and tested for the sensitivity to G418, hygromycin and puromycin, complete cell death was observed for each of the three drugs.…”

Section: Drug Resistance or Sensitivity In Mes1 Cellsmentioning

confidence: 99%

See 4 more Smart Citations

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

et al. 2004

Self Cite

View full text Add to dashboard Cite

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 93%

Section: Selectable Gene Cassettesmentioning

confidence: 99%

Section: Drug Selectionmentioning

confidence: 99%

Section: Drug Resistance or Sensitivity In Mes1 Cellsmentioning

confidence: 99%

See 3 more Smart Citations

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

et al. 2004

Self Cite

View full text Add to dashboard Cite

show abstract

Issues and Methodology for Development of Transgenic Fish for Aquaculture with a Focus on Growth Enhancement

Devlin

Raven

Sundström

et al. 2009

Molecular Research in Aquaculture

View full text Add to dashboard Cite

Fish ES Cells and Applications to Biotechnology

et al. 2006

Self Cite

View full text Add to dashboard Cite

ES cells provide a promising tool for the generation of transgenic animals with site-directed mutations. When ES cells colonize germ cells in chimeras, transgenic animals with modified phenotypes are generated and used either for functional genomics studies or for improving productivity in commercial settings. Although the ES cell approach has been limited to mice, there is strong interest for developing the technology in fish. We describe the step-by-step procedure for developing ES cells in fish. Key aspects include avoiding cell differentiation, specific in vitro traits of pluripotency, and, most importantly, testing for production of chimeric animals as the main evidence of pluripotency. The entire process focuses on two model species, zebrafish and medaka, in which most work has been done. The achievements attained in these species, as well as their applicability to other commercial fish, are discussed. Because of the difficulties relating to germ line competence, mostly of long-term fish ES cells, alternative cell-based approaches such as primordial germ cells and nuclear transfer need to be considered. Although progress to date has been slow, there are promising achievements in homologous recombination and alternative avenues yet to be explored that can bring ES technology in fish to fruition.

show abstract

Development of a positive–negative selection procedure for gene targeting in fish cells

Cited by 19 publications

References 29 publications

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

Issues and Methodology for Development of Transgenic Fish for Aquaculture with a Focus on Growth Enhancement

Fish ES Cells and Applications to Biotechnology

Contact Info

Product

Resources

About