Caution! Analyze transcripts from conditional knockout alleles

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Conditional knockout alleles are widely used to study gene function.
• Flanking exons with loxP sites (flox) and Cre-mediated recombination is a common strategy.
• Genomic DNA analysis is often considered sufficient to confirm knockout allele function.

Purpose of the Study:

• To evaluate a previously reported conditional knockout allele for the beta-subunit of protein farnesyltransferase.
• To investigate the necessity of RNA analysis in characterizing knockout alleles.
• To highlight potential pitfalls in interpreting knockout allele function based solely on DNA recombination.

Main Methods:

• Analysis of genomic DNA to confirm Cre-mediated recombination.
• mRNA sequencing and analysis to characterize the resulting transcripts.
• Prediction of protein products based on identified transcripts.

Main Results:

• Excision of exon 3 in the protein farnesyltransferase allele was confirmed at the DNA level.
• However, mRNA analysis revealed that exon 4 was skipped, leading to a "Deltaexon 3-4 transcript".
• This transcript results in a short in-frame deletion, not a frameshift, potentially yielding a partially functional protein.

Conclusions:

• Characterizing conditional knockout alleles requires both DNA and RNA level analysis.
• Relying solely on genomic DNA recombination can lead to erroneous conclusions about gene function.
• The protein farnesyltransferase allele serves as a critical example for ongoing mouse mutagenesis efforts.