Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background

Abstract

In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knockout"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly straindependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed.

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

“…There are several factors that have been demonstrated to affect the ES cell’s ability to transmit their genetic material through the germline including the genetic background, stemness, normality of karyotype, pathogen status of the ES cell line as well as the genetic background of recipient embryos [7], [21]. Among these factors, the combination of the genetic background of the ES cells and the recipient embryos has been demonstrated to be a critical factor affecting the germline transmissibility of the ES cells [7], [21], [22]. Ideally, the host background should allow the ES cells to have an optimal developmental advantage when injected into the blastocyst.…”

Germline Transmission of a Novel Rat Embryonic Stem Cell Line Derived from Transgenic Rats

“…The genetic background of recipient embryos also affects the germline transmissibility of ES cells [13][14][15]. Ideally, the host background should allow the ES cells to have an optimal developmental advantage when injected into the blastocyst.…”

“…Several factors, such as the genetic background, stemness, normality of karyotype, pathogen status of the ES cell line, as well as the genetic background of recipient embryos, affect the ES cells' ability to transmit their genetic material through the germline [13,14]. The utmost test for germline transmission is to inject the ES cell lines into recipient blastocysts to produce chimeric animals and then breed these animals to look for resulting pups with the ES cell genetic background.…”

“…Therefore, it is advantageous to prescreen and exclude cell lines that exhibit characteristics that might negatively impact their germline transmissibility. The ability of an ES cell line to be germline competent is affected by various factors, including the genetic background of the ES cell line, normality of its karyotype, passage number, cell morphology/ density, and pathogen status of the cell line including Mycoplasma status [14]. The genetic background of recipient embryos also affects the germline transmissibility of ES cells [13,15].…”

New and old technologies for organ replacement

“…As teratoma cells [22], embryonic stem cells [39], stem cells cloned by nuclear transfer [26,40], and induced pluripotent stem cells [30,39] can generate whole animals with full-sized, functioning organs, this challenge should be surmountable. Pluripotent stem cells, originated from fibroblasts, have been used to generate enough insulin-secreting cells with sufficient function to reverse hyperglycemia in mice with type 2 [41] and type 1 [42] diabetes.…”