Development of a retroviral strategy that efficiently creates nested chromosomal deletions in mouse embryonic stem cells and its exploitation for functional genomics.

摘要

Engineered nested chromosomal deletions are a valuable tool to explore the mouse genome functionalities, because they allow the examination of both protein-coding and non protein-coding regions. Up to now however, the generation of precisely localizable chromosomal deletions was laborious, precluding large-scale applications. The work presented in this thesis brings on anew alternative method to create genome-wide chromosomal deletions within a reasonable timeframe and applicable to both primary cells and to cell lines. This system relies on the creation of two compatible retroviruses delivering loxP sequences in the genome, the substrates required to perform Cre-induced site-specific recombination. The first section of this thesis (chapter 2) describes the strategy and the development of optimal retroviral vectors that were created to produce haploid chromosomal deletions in mouse embryonic stem cells. These engineered pluripotent cells revealed three haploinsufficient regions required for their proper in vitro differentiation and in vivo contribution to chimeric mice. These experiments validated the principles of this approach. The second section (chapter 3) provides the first large-scale exploitation of this new methodology. This involved the creation of a library of more than 1200 embryonic stem cell clones containing potential nested chromosomal deletions, localized throughout the mouse genome. The embryonic stem cell clones were used to perform functional screens and preliminary results uncovered numerous haploinsufficient regions that will be validated shortly. The retroviral constructs the engineered embryonic stem cell lines and their related functional annotations will be accessible to the scientific community within the coming year.;Keywords: chromosomal deletions, retroviruses, Cre-loxP, embryonic stem cells, functional genomics