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Multifunctional Alleles: A novel method for the generation of “All-In-One” null and conditional alleles

Author:
Pefanis, Evangelos, Schoenherr, Christopher, Persaud, Trikaldarshi, Zhang, Yuhong, Golubov, Jacquelynn, Wang, Bei, Skokos, Dimitris, Zambrowicz, Brian, Murphy, Andrew J., Economides, Aris N.
Source:
Methods 2019 v.164-165 pp. 91-99
ISSN:
1046-2023
Subject:
drugs, engineering, gene targeting, introns, null alleles, reporter genes, site-specific recombination
Abstract:
The engineering of conditional alleles has evolved from simple floxing of regions of genes to more elaborate methods. Previously, we developed Conditional by Inversion (COIN), an allele design that utilizes an exon-splitting intron and an invertible genetrap-like module (COIN module) to create null alleles upon Cre-mediated inversion. Here we build upon COINs by generating a new Multifunctional Allele (MFA), that utilizes a single gene-targeting step and three site-specific recombination systems, to generate four allelic states: 1. The initial MFA (generated upon targeting) functions as a null with reporter (plus drug selection cassette) allele, wherein the gene of interest is inactivated by both inversion of a critical region of its coding sequence and simultaneous insertion of a reporter gene. MFAs can also be used as ‘reverse-conditional’ alleles as they are functionally wild type when they are converted to COIN alleles. 2. Null with reporter (minus drug selection cassette), wherein the selection cassette, the inverted critical region, and the COIN module are removed. 3. COIN-based conditional-null via removal of the selection cassette and reporter and simultaneous re-inversion of the critical region of the target. 4. Inverted COIN allele, wherein the COIN allele in turn is reconverted to a null allele by taking advantage of the COIN module’s gene trap while simultaneously deleting the critical region.
Agid:
6394620