A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences.

Quantitative trait analysis may shed light on mechanisms regulating hematopoiesis in vivo. Strain-dependent variation existed among C57BL/6 (B6), DBA/2, and BXD recombinant inbred mice in the responsiveness of primitive progenitor cells to the early-acting cytokines kit ligand, flt3 ligand, and thrombopoietin. A significant quantitative trait locus was found on chromosome 2 that could not be confirmed in congenic mice, however, probably because of epistasis. Because it has been shown that alleles of unknown Xlinked genes confer a selective advantage to hematopoietic stem cells in vivo in humans and in cats, we also analyzed reciprocal male D2B6F1 and B6D2F1 mice, revealing an X-linked locus regulating the responsiveness of progenitor and stem cells to early-acting factors. Among DBA/2, B6, and BXD recombinant inbred mice, correlating genetic variation was found in the absolute number and frequency of Lin ؊ Sca1 ؉؉ kit ؉ cells, which are highly enriched in hematopoietic progenitor and stem cells, and in the number of Lin ؊ Sca1 ؉؉ kit ؊ cells, a population whose biologic significance is unknown, suggesting that both populations are functionally related. Suggestive quantitative trait loci (QTLs) for the number of Lin ؊ Sca1 ؉؉ cells on chromosomes 2, 4, and 7 were confirmed in successive rounds of mapping. The locus on chromosome 2 was confirmed in congenic mice. We thus demonstrated genetic variation in the response to cytokines critical for hematopoiesis in vivo and in the pool size of cells belonging to a phenotype used to isolate essentially pure primitive progenitor and stem cells, and we identified loci that may be relevant to the regulation of hematopoiesis in steady state. (Blood. 2002;99:3947-3954)

Current treatments of sporadic Burkitt's lymphoma (sBL) are associated with severe toxicities. A better understanding of sBL formation would facilitate development of less toxic therapies. The etiology of sBL remains, however, largely unknown, C-MYC up-regulation being the only lesion known to occur in all sBL cases. Several studies examining the role of C-MYC in the pathogenesis of BL have concluded that C-MYC translocation is not the only critical event and that additional unidentified factors are expected to be involved in the formation of this tumor. We herein report that a gene distinct from C-MYC, E2F1, is involved in the formation of all or most sBL tumors. We found that E2F1 is highly expressed in Burkitt's lymphoma cell lines and sBL lymphoma specimens. Our data indicate that its elevated expression is not merely the consequence of the presence of more cycling cells in this tumor relative to other cell lines or to other neoplasias. In fact, we show that reduction of its expression in sBL cells inhibits tumor formation and decreases their proliferation rate. We also provide data suggesting that E2F1 collaborates with C-MYC in sBL formation. E2F1 expression downregulation did not affect, however, the proliferation of human primary diploid fibroblasts. Because E2F1 is not needed for cell proliferation of normal cells, our results reveal E2F1 as a promising therapeutic target for sBL. [Cancer Res 2009;69(9):4052-8]