on Thursday, March 1st, 2018 1:41 | by Ottavia Palazzo
How the technique works: tRNA based vectors for producing multiple CRISPR sgRNA from a single transcript. Using multiple sgRNA will augment gene repression. In this technique the endogenous tRNA processing machinery liberates multiple functional sgRNA from a single precursor transcript in the nucleus. The vector used (pCFD6) permits cloning of several (three in this case) sgRNA flanked by Drosophila tRNA downstream of a single promoter.
The conditional KO can be done by using UAS-tRNA-sgRNA constructs.
PCR amplification product of three different FoxP fragments, those fragment will be ligated in only one appropriate vector.
PCR of the tentative of ligation of all the 3 fragments above: green arrows indicate clones that should be having incorporated all 3 fragment, orange arrows indicate clones that can be positive to all of 3 fragments but have a strange double band (the sequencing step will tell us the results).
Protocol for mice immunization for creating monoclonal Ab