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In-vitro Mutagenesis01:16

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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.
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TaqMan Multiplex qPCR Method to Genotype PARG Knockout Mice.

Yaroslava Karpova1,2, Alexei V Tulin3

  • 1Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA. iaroslava.karpova@und.edu.

Methods in Molecular Biology (Clifton, N.J.)
|December 14, 2022
PubMed
Summary

This study presents a new Taqman qPCR method for genotyping PARG knockout mice, crucial for studying poly(ADP-ribose) glycohydrolase (PARG) function. This technique enables precise genetic analysis, even with limited DNA, aiding research into this understudied enzyme.

Keywords:
Conditional knockoutLacZ genotypingMultiplex qPCRPARGPARPPoly(ADP-ribosyl)altion

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Area of Science:

  • Biochemistry
  • Genetics
  • Molecular Biology

Background:

  • Poly(ADP-ribose) (pADPr) synthesis is mediated by the PARP family, with over 20,500 publications.
  • PARG, the primary enzyme degrading pADPr, has significantly less research (around 500 papers) due to experimental challenges.
  • Difficulties include early embryonic lethality in PARG knockout animals and a lack of specific mouse antibodies.

Purpose of the Study:

  • To develop and present a reliable genotyping method for existing PARG knockout mice.
  • To enable efficient genetic analysis using small DNA amounts, including early embryos.
  • To facilitate research on PARG function and conditional knockout models.

Main Methods:

  • A Taqman qPCR multiplex approach was utilized for genotyping.
  • The method is designed to work with minimal DNA material.
  • It incorporates features to distinguish maternal DNA contamination.

Main Results:

  • A validated Taqman qPCR method for genotyping PARG knockout mice was established.
  • The assay allows for the analysis of early embryonic stages.
  • The method demonstrated its ability to separate maternal DNA contamination.

Conclusions:

  • The developed Taqman qPCR method provides a robust tool for PARG research.
  • This technique supports the study of PARG knockout and conditional knockout models.
  • It overcomes key experimental hurdles, promoting further investigation into PARG's role.