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Related Concept Videos

In-vitro Mutagenesis01:16

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
Lethal Alleles02:41

Lethal Alleles

Agouti: A Lethal Allele
Lucien Cuénot discovered lethal alleles in 1905 while studying the inheritance of coat color in mice. The agouti gene is responsible for the color of the coat in mice. This gene codes for an agouti-signaling protein, which is responsible for melanin distribution in mammals. The wild-type allele gives rise to gray-brown coat color in mice, while the mutant allele gives rise to yellow coat color. In addition to coat color, the agouti gene is associated with the yellow...
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Nonsense-mediated mRNA Decay

The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
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In Vivo Modeling of the Morbid Human Genome using Danio rerio
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Caution! Analyze transcripts from conditional knockout alleles.

Shao H Yang1, Martin O Bergo, Emily Farber

  • 1Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.

Transgenic Research
|December 19, 2008
PubMed
Summary
This summary is machine-generated.

Conditional knockout alleles require RNA analysis, not just DNA, to confirm gene disruption. A protein farnesyltransferase allele study revealed unexpected splicing, leading to a partial deletion instead of a null allele.

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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.