A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot.
Transgenic Res
; 26(3): 429-434, 2017 06.
Article
em En
| MEDLINE
| ID: mdl-28105543
ABSTRACT
The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific genomic locus, but limits the detection of its correct genomic integration by standard PCR methods. Genomic Long Range PCR (LR-PCR), using primers adjacent to the homology arms, has been used as an alternative to radioactive-based Southern blot screenings. However, LR-PCRs are often difficult and render many false positive and false negative results. Here, we propose an alternative screening method based on the detection of a genetic modification at the mRNA level, which we successfully optimized in two mouse models. This screening method consists of a reverse-transcription PCR (RT-PCR) using primers that match exons flanking the targeting construct. The detection of the expected modification in this PCR product confirms the integration at the correct genomic location and shows that the mutant mRNA is expressed. This is a simple and sensitive strategy to screen locus-specific recombination of targeting constructs which can also be useful to screen KO and KI mutant mice or cell lines including those generated by CRISPR/Cas9.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Recombinação Genética
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
/
Células-Tronco Embrionárias
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article