Abstract
We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse←rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction combined with intracytoplasmic sperm injection (TESE-ICSI). In the present paper, we disrupted rat hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene in ES cells and produced a Hprt-disrupted rat line using the mouse←rat ES chimera system. The mouse←rat ES chimera system demonstrated the dual advantages of space conservation and a clear indication of germ line transmission in knockout rat production.
Highlights
We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm
All six established transgenic rat lines F344-Tg (CAG/Acr3-EGFP) which we abbreviated as GBGS had ubiquitous fluorescence signal from somatic cells and testicular spermatozoa (Fig. 1 and Supplementary Fig. S1)
Lesch-Nyhan syndrome (LNS) is known to be caused by a mutation of the HPRT gene leading to a deficiency or complete absence of HPRT enzyme activity
Summary
We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). Animal and space requirements were identical to those for the gene disruption experiment in the mice This enabled an enhanced experimental scale, in case the efficiency of GLT of rat ES cells was low. Both mouse and rat spermatozoa were produced in the chimeric testes. The shape of the sperm heads differ in rats and mice, and the utilization of GFP facilitated selection of rat spermatozoa and subsequent microinsemination[13,14] With these strategies in mind, we chose hypoxanthine-guanine phosphoribosyl transferase (Hprt) as a target gene and sought to demonstrate the new method to produce a gene-disrupted rat line using the mouse← rat ES chimera system. One purpose of this study was to examine whether Hprt gene disruption in rats evokes self-injurious behaviour (SIB), as rats are better suited to behavioural studies and some drugs induce SIB in rats but not in mice[15]
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