Derivation of a Germline Competent Transgenic Fischer 344 Embryonic Stem Cell Line

Abstract

Embryonic stem (ES) cell-based gene manipulation is an effective method for the generation of mutant animal models in mice and rats. Availability of germline-competent ES cell lines from inbred rat strains would allow for creation of new genetically modified models in the desired genetic background. Fischer344 (F344) males carrying an enhanced green fluorescence protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. After establishment of ES cell lines, rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing was conducted in selected ES cell lines. One male ES cell line, F344-Tg.EC4011, was further evaluated for germline competence by injection into Dark Agouti (DA) X Sprague Dawley (SD) blastocysts. Resulting chimeric animals were bred with wild-type SD mates and germline transmissibility of the ES cell line was confirmed by identification of pups carrying the ES cell line-derived EGFP transgene. This is the first report of a germline competent F344 ES cell line. The availability of a new germline competent ES cell line with a stable fluorescence reporter from an inbred transgenic rat strain provides an important new resource for genetic manipulations to create new rat models.

Efficient generation of selection‐gene‐free rat knockout models by homologous recombination in ES cells

“…In our study, the frequency of both Leptin and p53 gene targeting via homologous recombination was 20% with SD and F344 ES cells, in comparison with previously reported typical efficiencies of 0.6-4.4% [27,34]. Although germline-competent rat ES cells have been derived from various strains [1,2,23,35], the knockout rats generated via homologous recombination have only been achieved in DA rats [27,32,34]. It has also been reported that the gene targeting event resulted in the failure of germline transmission in SD rat ES cells [32].…”

“…Although germline‐competent rat ES cells have been derived from various strains 1, 2, 23, 35, the knockout rats generated via homologous recombination have only been achieved in DA rats 27, 32, 34. It has also been reported that the gene targeting event resulted in the failure of germline transmission in SD rat ES cells 32.…”

“…The SD and F344 ES cells were obtained from Rat Resource and Research Center (SD‐Tg (GFP) 2BalRrrc‐ES1/Rrrc, RRRC#: 561; F344‐Tg (UBC‐EGFP) F455Rrrc‐ES4011/Rrrc, RRRC#: 654) 23, 24. All ES cells were cultured in 3i/Lif medium, supplemented with 0.5 m A83‐01 (Tocris, San Diego, CA, USA), 3 m m CHIR99021 (Selleck Chemicals, Houston, TX, USA), 0.5 m m PD0325901 (Selleck Chemicals), and 1000 units·mL −1 rat Lif (ESGRO, Millipore, Bedford, MA, USA) in N2B27 (Invitrogen, Carlsbad, CA, USA) medium.…”

2015 Guidelines for Establishing Genetically Modified Rat Models for Cardiovascular Research

“…However, considering the above limitations in efficiency, size of engineered knockins, and potential for intellectual property issues and scalability, the rat ESC still promises to have a significant and important role in the full potential of the rat model for CVD research. First established in 2008, authentic germline-competent rat ESC have now been derived from a few inbred, outbred, and hybrid strains, including Dark Agouti, Fisher F344, and Sprague Dawley [40, 45, 74–76]. These cell lines are also currently available through the RRRC.…”

Electroejaculation of chimeric rats

“…We carried out electroejaculation and semen collection for 11 male chimeric rats derived from injection of F344-Tg.EC4011/Rrrc ESCs (Rat Resource and Research Center RRRC# 654, Columbia, MO) into dark agouti × Sprague-Dawley blastocysts 7 . Rats were approximately 9–10 months of age.…”