METHODS OF MAKING TRANSGENIC MICE

Pronuclear Microinjection And Modifying Embryonic Stem Es Cells

Pronuclear Microinjection And Modifying Embryonic Stem Es Cells

Embryonic Stem Cells

Mouse embryonic stem (ES) cells are the in vitro counterparts of an in vivo population of cells, known as the epiblast, that are specific to the early embryo [1, 2 and 3]. Epiblast cells are pluripotent, which means that an individual cell can give rise to all cell types of the foetus. ES cells retain the developmental identity and potential of the epiblast even after prolonged culture. This has been shown conclusively by their complete integration into a developing embryo after being reintroduced into the blastocyst [4]. ES cells can efficiently colonize the germ line, resulting in chimaeric animals. These produce functional gametes, which allows ES cells to be used as vehicles for introducing sophisticated genetic modifications into mice . ES cells can also undergo multilineage differentiation in vitro and produce a range of well-differentiated progeny. Currently there is considerable interest in the prospect of exploiting this potential in analogous human pluripotent cells to generate specific, differentiated types of cell for drug development, for therapies based on cell replacement, and for delivering gene therapies.

Less attention has been paid to the unusual proliferative properties of ES cells. ES cells are derived without the intervention of any immortalizing agent, do not undergo either crisis or senescence, and retain a diploid karyotype. They proliferate without apparent limit and can readily be propagated clonally. They can multiply in the absence of serum and are not subject to contact inhibition or anchorage dependence. In fact there is no known means of inducing cell -cycle arrest and quiescence in ES cells. Apart from the normal karyotype, these are features that are typical of transformed cells and, indeed, ES cells are tumorigenic. In contrast to their behaviour when introduced into the early embryo, they produce teratocarcinomas when injected into adult mice, Thus ES cells can be considered as conditional tumour cells.

Cytokine-dependent activation of STAT3 drives ES cell self-renewal

The propagation of mouse ES cells is dependent on the presence of leukaemia inhibitory factor (LIF) or related cytokines that can activate signal transduction from cell-surface receptors . LIF can be provided by a feeder layer of embryonic fibroblasts [16 and 17] and/or as a recombinant protein. LIF engages a heterodimeric receptor complex consisting of two related cytokine receptors, LIF receptor (LIFR) and gp130 [18]. This complex activates associated Janus-associated (JAK) tyrosine kinases that phosphorylate the receptor chains. The phosphorylated tyrosines then act as docking sites for proteins containing Src homology 2 (SH2) domains that might themselves be phosphorylated by the JAKs ( Fig. 1).

Fig. 1. LIF-dependent activation of STAT3 blocks ES cell differentiation and promotes self-renewal. Leukaemia inhibitory factor (LIF) stabilizes the association of LIFR and gp130 cytokine receptors. Resultant activation of receptor-associated JAK kinases causes the recruitment, tyrosine phosphorylation and dimerization of STAT3. The STAT3 dimers then translocate to the nucleus, where they control the transcription of genes regulating ...