giuseppe testa - current research.

The broader goal of my work has been the development of improved strategies to engineer the mouse genome in embryonic stem (mES) cells, aiming at establishing faithful, clinically relevant models of human diseases.

Technologies to engineer intentional, heritable changes in the genomes of all life forms, whether prokaryotic or eukaryotic, are advancing rapidly. The availability of the whole sequence of the human genome offers an unprecedented opportunity to dissect the genetic basis of disease. However, in order to thoroughly understand molecular pathogenesis, the function and/or malfunction of relevant genes must be analysed in an in vivo model, which closely recapitulates human physiology. The experimental advantages of the mouse, through an unrivalled ability to engineer its genome in ES cells, has made it the premier model organism for studies of mammalian physiology and pathology. Until recently, engineering the mouse genome has been constrained by the limitations of the conventional DNA engineering methodologies used to construct transgenes or targeting vectors in E.coli. The development and application of homologous recombination strategies in E.coli, called ‘recombineering’, solved this problem so that now large cloned DNA molecules, such as BACs (bacterial artificial chromosomes), can be accurately engineered in E. coli (Yang et al., 1997; Zhang et al., 1998; Zhang et al., 2000) (Copeland et al., 2001) and then transferred into the mouse genome.

I have developed a strategy that simplifies the engineering of changes in the mouse genome. This simpler strategy is based on precise engineering of a cloned piece of the mouse genome in E. coli, and then introducing this engineered piece into the mouse genome. Consequently, the difficult task is accomplished using the convenience of E. coli.

Using Red/ET recombination (Muyrers et al., 1999; Zhang et al., 1998), I engineered complex changes into a large piece of mouse genomic DNA. Then, this large fragment was used to introduce a complex array of genetic changes in mouse ES cells (Testa, 2003). Specifically, bacterial artificial chromosomes (BACs) were engineered to generate large targeting constructs, which carry two selectable cassettes at the two ends and permit, in a single round of mouse embryonic stem (ES) cell transfection, simultaneous mutation of a given locus or target gene at two sites separated by considerable distances (> 40 kb). By flanking the selectable markers with site specific recombination target sites (SSRTs), for example loxP or FRT sites, these constructs generate multipurpose alleles. Hence a single ES cell clone can be used to derive a series of mutant mouse lines to address a range of biological questions. Since construct assembly and ES cell targeting are the current bottlenecks in mouse mutagenesis, approaches such as the one I developed are very relevant to streamline the use of the mouse as a model system and to fully harness the availability of its genome.

References

- Copeland, N. G., Jenkins, N. A., and Court, D. L. (2001). Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2, 769-779.
- Muyrers, J. P., Zhang, Y., Testa, G., and Stewart, A. F. (1999). Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res 27, 1555-1557.
- Testa, G., Zhang, Y., Vintersten, K., Benes, V., Pijnappel, W.W.M.P., Chambers, I., Smith, A.J.H., Smith. A.G. and Stewart, A.F. (2003). Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nature Biotechnology 21(4): 443-7.
- Yang, X. W., Model, P., and Heintz, N. (1997). Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol 15, 859-865.
- Zhang, Y., Buchholz, F., Muyrers, J. P., and Stewart, A. F. (1998). A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20, 123-128.
- Zhang, Y., Muyrers, J. P., Testa, G., and Stewart, A. F. (2000). DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol 18, 1314-1317.