Embryo technology in laboratory animals

Recent progress in genetics has been in part due to progress in embryo technology, as new developments in embryology have made increasingly sophisticated genetic manipulations possible. Although post-implantation embryos may in somecases be used as experimental subjects. most procedures make use of pre-implantation stages. Techniques such as in Vitro culture and fertilisation of embryos, embryo transfer and the cryopreservation of gametes and embryos are now routinein many laboratories. and play an important part in the creation of genetically manipulated animals. The production of chimaeras has also become routine. Aggregation chimaeras have been made for some years‘ while injection chimaeras (created by the injection of embryonic stem cells or blastomeres into the blastocyst cavity) play an important part in the production of mice with alterations o specific genes (the process of "gene targeting").The techniques of embryo cloning andembryo biopsy have also found practical applications in animal production and medicine. But one of the most significant advances in embryo technology has been the development of embryonic stem cells (ES cells), lines of cultured cells thatcan, when introduced into a host blastocyst, participate in the formation of all tissues. Animals can thus be produced carrying genetic alterations induced in ES cells. Although at present ES cells are available for only a few strains of mice, it is expected that in the future ES cell lines from other species will be developed

Efficient Gene Targeting by Homologous Recombination in Rat Embryonic Stem Cells

The rat is the preferred experimental animal in many biological studies. With the recent derivation of authentic rat embryonic stem (ES) cells it is now feasible to apply state-of-the art genetic engineering in this species using homologous recombination. To establish whether rat ES cells are amenable to in vivo recombination, we tested targeted disruption of the hypoxanthine phosphoribosyltransferase (hprt) locus in ES cells derived from both inbred and outbred strains of rats. Targeting vectors that replace exons 7 and 8 of the hprt gene with neomycinR/thymidine kinase selection cassettes were electroporated into male Fisher F344 and Sprague Dawley rat ES cells. Approximately 2% of the G418 resistant colonies also tolerated selection with 6-thioguanine, indicating inactivation of the hprt gene. PCR and Southern blot analysis confirmed correct site-specific targeting of the hprt locus in these clones. Embryoid body and monolayer differentiation of targeted cell lines established that they retained differentiation potential following targeting and selection. This report demonstrates that gene modification via homologous recombination in rat ES cells is efficient, and should facilitate implementation of targeted, genetic manipulation in the rat

Experimental approaches to establish rat embryonic stem cells

The rat has been an established experimental animal model within many areas of biological investigation for over one hundred years due to its size, breeding characteristics, and knowledge of its physiology and behaviour. In recent years its status as a leading biomedical model has been somewhat surpassed by the mouse. This is largely the result of the isolation and application of mouse embryonic stem (ES) cells. Mouse ES cells have the capacity for unlimited self-renew in vitro whilst maintaining pluripotency and germline competence, and most importantly are amenable to sophisticated reverse genetics strategies such as gene targeting, which have provided a route to germ line modification. Thus far, the derivation of rat ES cells has proved elusive. The generation of rat ES cells would therefore facilitate equivalent applications to rat genetics and significantly strengthen the rat as an experimental model system. Previous attempts to derive rat ES cells led to the isolation of rat ES-like cells. However, whilst these cells exhibit extensive self-renew in vitro, it was known that they fail to maintain significant levels of the key functional ES cell marker Oct4 and do not contribute to chimeras. Rather, these cells express the trophectoderm markers Cdx2 and CyclinD3, and have been termed ExS cells due to their probable extra-embryonic nature. In the work described in this thesis, further investigation of ExS cells revealed the absence of expression of the key pluripotency gene Nanog, although the expression pattern of Nanog in the rat embryo was shown to be similar to that of mouse. It was hypothesised that expression of exogenous Oct4 and Nanog or Sox2 genes could facilitate reprogramming of ExS cells into a 'true' ES cell state. Initial work described in this thesis demonstrated that it was possible to introduce transgenes into rat ExS cells and obtain stable transformants with long term transgene expression. On this basis Oct 4, Nanog and Sox2 transgene expression vectors were constructed and stably integrated into ExS cells, and transgene expression verified. However, no reactivation of an endogenous gene expression profile, characteristic of a true ES cell-like state, was observed in any of the transgenic lines produced. Concurrent with work on ExS cells, investigations by others using chemically defined, serum-free medium containing small molecule inhibitors of MEK and GSK3 (called 3i/2i medium) had demonstrated that it was possible to readily isolate mouse ES cells, even from strains known to be refractory to ES cell isolation. Therefore, the ability of this culture system to facilitate rat ES cell derivation was investigated. Rat 3i/2i cell lines were established from ICM outgrowths of Fischer, DA and Sprague Dawley E4.5 rat embryos. These cells maintained expression of Oct4 and Nanog and could generate complex teratomas consisting of all three germ layers. They were distinct from epiblast stem cells (EpiSC) in that they expressed Klf4, Rex1 and Stella and most importantly, they could contribute to the formation of adult chimaeras and demonstrated germline competency. Isolation of these authentic rat ES cells paves the way for gene targeting in the rat, a development that should greatly facilitate new biomedical discoveries.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Capture of Authentic Embryonic Stem Cells from Rat Blastocysts

SummaryEmbryonic stem (ES) cells have been available from inbred mice since 1981 but have not been validated for other rodents. Failure to establish ES cells from a range of mammals challenges the identity of cultivated stem cells and our understanding of the pluripotent state. Here we investigated derivation of ES cells from the rat. We applied molecularly defined conditions designed to shield the ground state of authentic pluripotency from inductive differentiation stimuli. Undifferentiated cell lines developed that exhibited diagnostic features of ES cells including colonization of multiple tissues in viable chimeras. Definitive ES cell status was established by transmission of the cell line genome to offspring. Derivation of germline-competent ES cells from the rat paves the way to targeted genetic manipulation in this valuable biomedical model species. Rat ES cells will also provide a refined test-bed for functional evaluation of pluripotent stem cell-derived tissue repair and regeneration