|25 May 2009
||11:00am | Breakthrough Theatrette, Level 4, Matrix.
||Prof. Allan Bradley
|Genetic Screens in Embryonic Stem Cells
Prof. Allan Bradley, Director of Wellcome Trust Sanger Institute, Hinxton, U.K.
Of the estimated 23,000 genes identified in the human and mouse genomes, the majority lack functional annotation. Mutations can now be generated extremely rapidly and with nucleotide precision in ES cells. However, the analysis of each mutant requires transmitting an allele through the mouse germ line followed by two generations of breeding to generate homozygous mutant individuals. Although very informative about gene function, this approach can only be performed on a small scale and is thus limited.
In principle, cultured embryonic stem cells (ES) provide access to the function of a significant fraction of genes in the genome. The expressed genome of un-differentiated ES cells comes from at least 10,000 genes, whose products are required to elaborate the fundamental components required for a mammalian cell such as the structural components and physiological systems for essential functions like metabolism, cell division and DNA repair. ES cells can also be induced to differentiate, providing access to other types of genes such as those involved in signalling cascades and elaborating cell differentiation programmes.
Bi-allelic (homozygous) mutations have been generated in ES cells, but to date this has only been conducted on a gene-by-gene basis. Single allele insertional mutations can be efficiently induced using retroviral gene-trap vectors in ES cells. This approach has been used to establish extensive libraries of ES cells with mutations. However, these libraries are composed of heterozygous mutations thus they are not suitable for conducting recessive screens. By exploiting the high rate of mitotic recombination in Blm-deficient ES cells1, we have been able to increase the frequency of homozygous mutations in these libraries to 0.1-1.0% of the mutagenized pool. Screens conducted using these libraries have identified genes required for two different biological processes, namely DNA mismatch repair and retroviral infection2,3.
Mutations generated by gene-trap retroviruses are limited in their genome coverage because of integration bias. We have overcome this limitation by using the PiggyBac transposon to construct insertional gene-trap mutation libraries in Blm-deficient ES cells4. These mutation libraries have excellent genomic coverage, enabling the full repertoire of genes required for biological processes to be recovered from screens. In contrast to RNAi based approaches, PiggyBac libraries in Blm-deficient ES cells are simple to generate and the knockdown’s of each gene’s function is complete. This genetic system opens a new and powerful approach for conducting phenotype and genotype based recessive genetic screens in mammalian cells in culture.
1. Luo G, Santoro I, McDaniel LD, Nishijima I, Mills M, Youssoufian H, Vogel H, Schultz RA, Bradley A (2000) Cancer predisposition caused by elevated mitotic recombination in Bloom mice. Nature Genetics 26, 424-429.
2. Ge Guo, Wei Wang & Allan Bradley. (2004) Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells. Nature 429 891-895
3. Wang, W., Bradley, A. A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells Genome Biology 8(4):R48 (2007)
4. Wang, W., Bradley, A. & Huang, Y. (2009), "A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells", Genome research, vol. 19, no. 4, pp. 667-673
Host: Prof. Neal Copeland