Limits...
Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.

Li S, Li Z, Shu FJ, Xiong H, Phillips AC, Dynan WS - Nucleic Acids Res. (2014)

Bottom Line: We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes.Of 12 genes tested, none were downregulated, and several were upregulated.Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Emory University, Atlanta, GA 30322, USA Department of Biochemistry, Emory University, Atlanta, GA 30322, USA Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, GA 30912, USA.

Show MeSH

Related in: MedlinePlus

Derivation and validation of NONO-deficient MEFs. (A) Portion of mouse X chromosome depicting the Nono locus. Positions of gene trap within second intron and primers used for genotyping are shown. The mRNA product of the gene trap allele, which does not contain any of the Nono open reading frames, is shown in red. (B) Representative image showing genotyping by PCR. Left, analysis using primers P1 and P2 to detect wild-type allele; right, analysis using primers P3 and P4 to detect gene trap allele. In each panel, lane 1, wild-type male (+/0); lane 2, heterozygous female (gt/+); lane 3, Nono-gene trap male (gt/0). (C) Expression of NONO and β-geo gene trap mRNAs in tissues of mice sacrificed at postnatal day 4. Image shows PCR products with β-actin as a loading control. (D) NONO, SFPQ and internal reference protein (β-actin) expression in MEF isolates from mice bearing wild type (+) or gene trap (gt) alleles. Total cell lysates were probed with indicated antibodies. Left panel, gel image; right panel, quantification. (E) Immunostaining of the same four MEF isolates with anti-NONO antibody. Scale bar denotes 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150768&req=5

Figure 1: Derivation and validation of NONO-deficient MEFs. (A) Portion of mouse X chromosome depicting the Nono locus. Positions of gene trap within second intron and primers used for genotyping are shown. The mRNA product of the gene trap allele, which does not contain any of the Nono open reading frames, is shown in red. (B) Representative image showing genotyping by PCR. Left, analysis using primers P1 and P2 to detect wild-type allele; right, analysis using primers P3 and P4 to detect gene trap allele. In each panel, lane 1, wild-type male (+/0); lane 2, heterozygous female (gt/+); lane 3, Nono-gene trap male (gt/0). (C) Expression of NONO and β-geo gene trap mRNAs in tissues of mice sacrificed at postnatal day 4. Image shows PCR products with β-actin as a loading control. (D) NONO, SFPQ and internal reference protein (β-actin) expression in MEF isolates from mice bearing wild type (+) or gene trap (gt) alleles. Total cell lysates were probed with indicated antibodies. Left panel, gel image; right panel, quantification. (E) Immunostaining of the same four MEF isolates with anti-NONO antibody. Scale bar denotes 10 μm.

Mentions: The knockout mice used in these studies were genetically similar to those in a recent study (12) but were independently derived. They contain a gene trap insertion in the second intron in the X-linked Nono locus (Figure 1A). As the first two exons are noncoding, the predicted hybrid Nono- β-geo transcript does not contain any of the native Nono open reading frame. Mice were produced from the ES cells by standard techniques. DNA repair and radiosensitivity studies used mice that were backcrossed for >10 generations into the C57/Bl6 background.


Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.

Li S, Li Z, Shu FJ, Xiong H, Phillips AC, Dynan WS - Nucleic Acids Res. (2014)

Derivation and validation of NONO-deficient MEFs. (A) Portion of mouse X chromosome depicting the Nono locus. Positions of gene trap within second intron and primers used for genotyping are shown. The mRNA product of the gene trap allele, which does not contain any of the Nono open reading frames, is shown in red. (B) Representative image showing genotyping by PCR. Left, analysis using primers P1 and P2 to detect wild-type allele; right, analysis using primers P3 and P4 to detect gene trap allele. In each panel, lane 1, wild-type male (+/0); lane 2, heterozygous female (gt/+); lane 3, Nono-gene trap male (gt/0). (C) Expression of NONO and β-geo gene trap mRNAs in tissues of mice sacrificed at postnatal day 4. Image shows PCR products with β-actin as a loading control. (D) NONO, SFPQ and internal reference protein (β-actin) expression in MEF isolates from mice bearing wild type (+) or gene trap (gt) alleles. Total cell lysates were probed with indicated antibodies. Left panel, gel image; right panel, quantification. (E) Immunostaining of the same four MEF isolates with anti-NONO antibody. Scale bar denotes 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150768&req=5

Figure 1: Derivation and validation of NONO-deficient MEFs. (A) Portion of mouse X chromosome depicting the Nono locus. Positions of gene trap within second intron and primers used for genotyping are shown. The mRNA product of the gene trap allele, which does not contain any of the Nono open reading frames, is shown in red. (B) Representative image showing genotyping by PCR. Left, analysis using primers P1 and P2 to detect wild-type allele; right, analysis using primers P3 and P4 to detect gene trap allele. In each panel, lane 1, wild-type male (+/0); lane 2, heterozygous female (gt/+); lane 3, Nono-gene trap male (gt/0). (C) Expression of NONO and β-geo gene trap mRNAs in tissues of mice sacrificed at postnatal day 4. Image shows PCR products with β-actin as a loading control. (D) NONO, SFPQ and internal reference protein (β-actin) expression in MEF isolates from mice bearing wild type (+) or gene trap (gt) alleles. Total cell lysates were probed with indicated antibodies. Left panel, gel image; right panel, quantification. (E) Immunostaining of the same four MEF isolates with anti-NONO antibody. Scale bar denotes 10 μm.
Mentions: The knockout mice used in these studies were genetically similar to those in a recent study (12) but were independently derived. They contain a gene trap insertion in the second intron in the X-linked Nono locus (Figure 1A). As the first two exons are noncoding, the predicted hybrid Nono- β-geo transcript does not contain any of the native Nono open reading frame. Mice were produced from the ES cells by standard techniques. DNA repair and radiosensitivity studies used mice that were backcrossed for >10 generations into the C57/Bl6 background.

Bottom Line: We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes.Of 12 genes tested, none were downregulated, and several were upregulated.Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Emory University, Atlanta, GA 30322, USA Department of Biochemistry, Emory University, Atlanta, GA 30322, USA Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, GA 30912, USA.

Show MeSH
Related in: MedlinePlus