A Search for Genes Specifying HSCs through the Development of a New Retroviral-Based System That Creates Nested Deletions in ES cells.

With the intention of identifying novel DNA segments essential for hematopoietic stem cell (HSC) specification, we have taken advantage of retroviral-based delivery of loxP sequences to ES cells in order to create large genomic deletions. Cre-induced recombination between the integrated loxP-containing proviruses is selected based on the reconstitution of a functional neomycin resistance cassette. A unique set of complementary retroviruses (virus A and D) was finally identified after the generation and testing of more than 10 different cassettes. The nature of the rearrangements produced (deletions, inversions, etc.) was determined according to the sensitivity to selection marker genes incorporated inside the viruses and confirmed by Southern blot analysis of genomic DNA extracted from selected clones. The new technology was first tested on 11 different clones infected at a low MOI of virus D (to obtain one integrant per cell). This virus, selected under puromycin, contains also a neomycin resistance gene (neo^r), devoid of an initiation codon (ATG) and of a functional promoter. The proviral integration sites are now easily determined by inverse PCR using primers anchored at the neo^r gene. The second retrovirus (virus A), selected with hygromycin, integrates randomly another loxP site and a pgk promoter which precedes the ATG-loxP cassette. For the 11 primary clones tested to date, the number of secondary clones derived ranged between 7000 and 37 000. Recombination between the loxP sites, upon Cre addition, is selected by the functional reconstitution of the neomycin gene (i.e. pgk-ATG-loxP-neo^r). Out of 10⁷cells electroporated with the cre plasmid, neomycin resistant clones were obtained for each population (ranging between 7 and 188). Rearranged tertiary clones (i.e., virus D+A+Cre) derived from a selected primary clone (no.9) have been studied extensively by Southern Blot analysis. Chromosomal rearrangements correlated with bands of expected sizes (3.0 kb compare to 3.4 kb for the unrearranged primary and secondary clones, using KpnI digests and a neomycin probe). Clonal analysis for integration of the second retrovirus (virus A) revealed at least 13 different deletions out of 34 tested. In one experiment, very preliminary results suggest that 3 out of these 13 clones bearing independent deletions failed to differentiate upon LIF removal. Among the other 10 primary clones saturated to date, 9 gave tertiary clones sensitive to puromycin (undergoing selection). Overall, these results document that our novel retroviral-based strategy is amenable to functional screening of genes that specify HSC in differentiating ES cells. Moreover, this technology is efficient (11 out of 11 clones tested), rapid and applicable to any cellular system.