1. Multifunctional Alleles: A novel method for the generation of 'All-In-One' null and conditional alleles
- Author
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Dimitris Skokos, Brian Zambrowicz, Aris N. Economides, Yuhong Zhang, Trikaldarshi Persaud, Schoenherr Christopher, Jacquelynn Golubov, Evangelos Pefanis, Andrew J. Murphy, and Bei Wang
- Subjects
Hypoxanthine Phosphoribosyltransferase ,Genetic Vectors ,Primary Cell Culture ,Computational biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Mice ,03 medical and health sciences ,Genes, Reporter ,otorhinolaryngologic diseases ,Animals ,Coding region ,Allele ,Molecular Biology ,Alleles ,Floxing ,030304 developmental biology ,0303 health sciences ,Reporter gene ,030302 biochemistry & molecular biology ,Null (mathematics) ,Wild type ,Gene targeting ,Mouse Embryonic Stem Cells ,Exons ,Null allele ,Introns ,stomatognathic diseases ,Gene Targeting ,Genetic Engineering ,Interleukin Receptor Common gamma Subunit - 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.
- Published
- 2019
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