Establishment Of Fah^-/- Rat Embryonic Stem Cells

Gene Knockout has been a powerful gene functional research technique since developed during1980s, based on mammalian homologous recombination and embryonic stem cell related isolation and culture technology. Since the first gene knockout mouse model with hrpt deficient was established in1989by Thompson, bunch of gene knockout mouse models have emerged and been used in all aspects of biological field. But other bigger mammals like rat, pig are closer to human being in weight, pathology and other aspects, they have been considered as better models for their closer characteristics compared to mouse. And this led scientists put their endeavors into establishing bigger models when generating and using mouse models.Rat has been scientific animal for longer than150years, and since King generated the first inbred rat strain, more than500strains have been experimental available. But establishing rat embryonic stem cell has been a failure. Until2008, Ying reported his success in establishing rat embryonic stem cells, this reminded scientists of rat model again. Two years after this Ying made a headlined work by establishing P53knockout rat model. Elie Dolgin and F. kent Hamra highly appraised Ying’s work in Science and Nature respectively. This declares "rat gene knockout" age.Inspired by Ying’s work, we applied traditional homologous recombination method to knockout fah gene aiming at the establishment of fah knockout rat model (hereditary tyrosinaemia type I, HT1) in this study. We first extracted the DA ES cells from DA blastocytes and used both made in-house and given by Ying DA ES cells to conduct knockout trail. In molecular trials, we first constructed the versatile targeting vector in vitro with positive-negative selective cassette, and inserted targeting arms amplified by Phusion High-Fidelity PCR kit, then linearized the vector for future use. In cell trials, we successfully derived DA ES cells and related pluripotency factors were tested, results manifested our success. Then we transfered the linearized vector into ES cells through electroporation. And we used2i ES culture medium containing0.5μg/ml puromycin to select puromycin resistant clones48h after electroporation, after three cycles of selection, we picked24clones including3clones stably expressing enhanced green fluorescent protein at a higher rate than90%. PCR was then carried out to confirm targeted ES cell clones. And three fah deficient rat ES cell clones were successfully established, this facilitates and is a linchpin for the establishment of fah knockout rat model.