Dr Andrew Smith

Web Links:
Diagram explaining the principle of Recombinase-Mediated Genomic Replacement (RMGR).

Diagram explaining the principle of Recombinase-Mediated Genomic Replacement (RMGR).

GFP reporter expression in SOX-2 knock-in SHEF 4 human ES cell line. SHEF 4 human ES cells were created by gene targeting to insert a GFP reporter coding sequence with a linked IRES-puromycin resistance gene in frame at the ATG initiation codon of the SOX-2 gene. All cells show sustained high-level GFP fluorescence (right panel) when cultured in mTeSR medium supplemented with puromycin selection and grown on Matrigel matrix.

GFP reporter expression in SOX-2 knock-in SHEF 4 human ES cell line. SHEF 4 human ES cells were ...

A major advance has been the capability to make precise multi-kilobase genomic replacements in mouse ES cells with human sequence from Bacterial Artificial Chromosomes (BACs). This involved combining the technologies of recombineering in E. coli, with gene targeting by homologous recombination and site-specific recombination in ES cells. This strategy, called Recombinase-Mediated Genomic Replacement (RMGR), the principle of which is explained in Figure 1, allowed 'humanisation' of approximately 100 kilobases (kb) of the mouse α-globin locus encompassing the entire regulatory domain using a human BAC clone containing α-globin synteny region (see Figure 1).

The use of genetically modified mouse embryonic stem (ES) cells and transgenic animals derived from these underpins numerous investigations in biological and medical research. Fundamental understanding of gene function and regulation of gene expression, cell and developmental biology, and physiology has benefitted from the application of ES cell genetic engineering. Genetically modified mouse ES cells and transgenic animals also enable accurate models of specific human diseases to be created to further understanding of disease pathology and to explore potential therapeutic approaches. Therefore, new strategies that extend the range and complexity of genetic modifications in mouse ES cells to allow novel experimental approaches are desirable to enhance the success of future investigations.

In addition, genetic manipulation of human ES cell lines is essential to fully exploit the opportunities these can offer as an experimental platform. For example, in order to investigate events in human embryonic development, which are not otherwise accessible to experimental intervention, by genetic approaches and in-vitro differentiation; and to effectively implement these in applied biomedical projects such as drug discovery by use of genetically modified human ES cell lines. Therefore, an important goal is to achieve proficiency in human ES cell genetic manipulation equivalent to that presently attainable in mouse ES cells.

There are no collaborations listed for this principal investigator.

There are no publications listed for this principal investigator.