tag:blogger.com,1999:blog-32683882.post115619680792805523..comments2023-07-29T02:58:08.990-05:00Comments on BioMed Notes: Immunity: New T cell Regulatory ProteinsUnknownnoreply@blogger.comBlogger2125tag:blogger.com,1999:blog-32683882.post-1156197099474794352006-08-21T16:51:00.000-05:002006-08-21T16:51:00.000-05:00The most frequent clone they obtained (46 times) w...The most frequent clone they obtained (46 times) was a T cell receptor beta chain (TCRb, Table 1), which replaced the endogenous TCRb. The altered TCR was no longer triggered by the antibody, so CD69 was not increased. Tet turned off the new beta chain, allowing the old TCR to be expressed.<BR/><BR/>Any ideas why they didn't find TCR alpha clones? Is this chain expressed at higher levels (making it less easily competed by the transduced gene) or is it much more conserved (so that the antibody could still bind)?<BR/><BR/>---<BR/>What does not kill me makes me more susceptible to the next infection.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-32683882.post-1156197007805616682006-08-21T16:50:00.000-05:002006-08-21T16:50:00.000-05:00Useful Winnowing StepA key technical advantage was...Useful Winnowing Step<BR/><BR/>A key technical advantage was their use of a tetracylin-regulated ('Tet off') retroviral expression construct. The promoter controlling expression of the inserted cDNA contained a Tet-regulated element (TRE, Fig 1).<BR/><BR/>Tet regulation allowed them to quickly check the candidate T cell clones for artifactual (epigenetic) CD69 inhibition. If adding tetracyclin (thereby turning off expression of the transduced protein) did not restore CD69 induction, then the transduced protein was not responsible for the inhibition and the investigators could discard the clone. More than half the transductants showing decreased CD69 induction were NOT rescued by Tet treatment (Fig 2c), so this check saved significant effort by allowing them to focus on analyzing the 1,323 clones that passed this test out of 2,828 clones obtained initially (page 6).Anonymousnoreply@blogger.com