Efficient Integration of Short Interspersed Element-flanked Foreign DNA via Homologous Recombination

Abstract

We investigated whether mouse short interspersed elements (SINEs) could influence the recombination frequency of foreign DNA. Vectors harboring a reporter gene in combinations of SINEs B1 and/or B2 or a portion of long interspersed element-1 were prepared and tested in vitro by a colony assay using HC11 murine mammary epithelial cells and in vivo by microinjection into fetilized mouse eggs. In transfected HC11 cells, the number of colonies surviving G418 selection increased by 3.5-fold compared with control when the reporter was flanked by fused B1-B2 sequences. Similar results were obtained from microinjection study; in fetuses 11.5 days post coitum, transgene positives in control and SINEflanked vectors were 16 and 53%, respectively. Individual B1-and B2-harboring vectors showed equivalent activities with each other, as determined by the colony assay (2.8-fold versus 3.2-fold compared with control). We determined the contribution of homologous recombination to the SINE-mediated increase in integration frequency through a polymerase chain reaction-based strategy; in more than half of embryos transgenes underwent homologous recombinations involving B1 sequences. These results demonstrate that the SINE sequences can increase the integration rate of foreign DNA and that such an increase is most likely due to the enhancement of homologous recombination.

Chromosome integration of BAC (bacterial artificial chromosome): evidence of multiple rearrangements

“…Several works were designed to know whether this phenomenon can be influenced by the sequence of the injected DNA insert. The addition of SINEs elements (short interspersed elements) at both side of foreign DNA increased the efficiency of integration in mouse embryo genome, and this was probably due to the enhancement of homologous recombination (Kang et al 1999). In the present work, the long pig DNA fragments encompass series of repetitive sequence regions such as SINEs or LINEs.…”

The Histone H3K79 Methyltransferase Dot1L Is Essential for Mammalian Development and Heterochromatin Structure

“…DNA from the yolk sac was used for genotyping by PCR using the primers described above. X-gal staining was performed on 7.5- to 12.5-dpc Dot1L 3lox/+ embryos and littermates as previously described [30]. Embryo, yolk sac, and placental tissue specimens, which were harvested at 9.5-dpc, were fixed in Bouin's solution, washed extensively in 70% ethanol, processed routinely for paraffin embedding, sectioned at 5 µm, stained with hematoxylin and eosin, and then evaluated by bright field microscopy.…”

Dual Functions of Histone-Lysine N-Methyltransferase Setdb1 Protein at Promyelocytic Leukemia-Nuclear Body (PML-NB)

“…Unfertilized metaphase II stage oocytes were collected from ampullae of superovulated females without mating. Microinjection was performed 24 h after human chorionic gonadotropin injection (30). To visualize the pronuclei, cumulus-free zygotes were centrifuged at 13,000 rpm for 5 min.…”