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Total RNA was extracted and subjected to RT-PCR analysis (1/5 serial dilutions) for specific detection of Lsh or 18s (control) m RNA.
() Inhibition of Lsh m RNA by immunosuppressive drugs.
3 and 4; note that the genetic sequence has been entered in Genbank as Lsh (lymphoid specific helicase) and in the mouse genome database as Hells (helicase, lymphoid-specific)].
Lsh m RNA was found to be preferentially expressed in lymphoid tissue in the adult mouse, which contrasts with the ubiquitous expression of many other mammalian SNF2/helicase homologs (3, 4).
The thymus of 4–8 wk old mice was stained with anti-CD4-PE and anti-CD8-FITC and sorted into double negative, double positive, and single positive cell populations.
We recently cloned Lsh/Hells from thymocytes undergoing rearrangement, using a PCR-based strategy exploiting the highly conserved helicase domains [refs.In this study, we examined the expression pattern of Lsh m RNA and protein during lymphoid differentiation and activation.To test the role of Lsh in lymphoid development, we deleted the gene in mice.Several genes involved in the regulation of apoptosis were expressed normally with the exception of Bcl-2 that was actually elevated.These findings demonstrate that Lsh is not obligatory for normal lymphoid development but is essential for normal proliferation of peripheral T lymphocytes.
( Single cell suspensions from thymus, spleen, lymph nodes, and ficolled blood (lympholyte M, Cedarlane Laboratories) were stained and analyzed by flow cytometry using a FACStar Plus (Becton Dickinson).