Limits...
Scaffold attachment factor B2 (SAFB2)- mice reveal non-redundant functions of SAFB2 compared with its paralog, SAFB1.

Jiang S, Katz TA, Garee JP, DeMayo FJ, Lee AV, Oesterreich S - Dis Model Mech (2015)

Bottom Line: Scaffold attachment factors SAFB1 and SAFB2 are multifunctional proteins that share >70% sequence similarity.Further analysis showed a significantly increased testis weight in SAFB2(-/-) mice, which was associated with an increased number of Sertoli cells.Our data suggest that this is at least in part caused by alterations in androgen-receptor function and expression upon deletion of SAFB2.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

No MeSH data available.


Related in: MedlinePlus

Generation of SAFB2−/− mice. (A) SAFB2 mouse allele, and targeting construct for deletion of the SAFB2 genomic fragment from exons 4 to 10. SB, Southern blot probe. (B) Southern blot analysis of genomic DNA from embryonic stem (ES) cells (left panel) and mice (right panel). SAFB2+/+ and SAFB2−/− bands have sizes of 12 kb and 5 kb, respectively. (C) Northern blot analysis of RNA from SAFB2+/+, SAFB2+/− and SAFB2−/− mice. (D) RT-PCR using RNA from SAFB2+/+, SAFB2+/− and SAFB2−/− mice and primers targeting both the N and C terminal regions of SAFB2 and SAFB1. NTC, non-template control. (E) Immunoblot of lysates from either SAFB2+/+ or SAFB2−/− mice with antibodies targeting SAFB2 and SAFB1. Lower panel shows an entire SAFB2 immunoblot to provide proof of expression of the N-terminal fragment. (F) LacZ (β-galactosidase) staining of SAFB2+/+ or SAFB2−/− Sertoli cells. Blue, DAPI; red, Alexa Fluor 546 (lacZ).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4582101&req=5

DMM019885F1: Generation of SAFB2−/− mice. (A) SAFB2 mouse allele, and targeting construct for deletion of the SAFB2 genomic fragment from exons 4 to 10. SB, Southern blot probe. (B) Southern blot analysis of genomic DNA from embryonic stem (ES) cells (left panel) and mice (right panel). SAFB2+/+ and SAFB2−/− bands have sizes of 12 kb and 5 kb, respectively. (C) Northern blot analysis of RNA from SAFB2+/+, SAFB2+/− and SAFB2−/− mice. (D) RT-PCR using RNA from SAFB2+/+, SAFB2+/− and SAFB2−/− mice and primers targeting both the N and C terminal regions of SAFB2 and SAFB1. NTC, non-template control. (E) Immunoblot of lysates from either SAFB2+/+ or SAFB2−/− mice with antibodies targeting SAFB2 and SAFB1. Lower panel shows an entire SAFB2 immunoblot to provide proof of expression of the N-terminal fragment. (F) LacZ (β-galactosidase) staining of SAFB2+/+ or SAFB2−/− Sertoli cells. Blue, DAPI; red, Alexa Fluor 546 (lacZ).

Mentions: SAFB2−/− mice were generated by homologous recombination in 129/Sv ES cells, replacing exons 4-10 with an IRES-lacZ-SV40pA (β-galactosidase) marker and a PGK-Neomycin-bGHpA selection marker (Fig. 1A). The targeting construct was linearized and electroporated into ES cells, and the neomycin-resistant ES cell colonies were screened by Southern blot analysis (Fig. 1B, left panel). Three recombinant clones were injected into C57BL/6 blastocysts, and two (B11 and G12) were transmitted to germ line. The chimeric mice were bred with wild-type C57BL/6 mice to produce F1 mixed background (129/Sv and C57BL/6) heterozygous mutant mice (SAFB2+/−). Heterozygous F1 mice from each line were intercrossed to produce two independent lines of homozygous mutant mice (SAFB2−/−). Genotyping was confirmed by Southern blot analysis (Fig. 1B, right panel). Loss of SAFB2 mRNA expression was confirmed by northern blot analysis (Fig. 1C), and by RT-PCR assays using primers spanning the Safb2 C-terminus (exons 18-21) (Fig. 1D). RT-PCR with primers covering exons 4-7 revealed remaining expression of the N-terminus. SAFB1 mRNA expression was not affected, as shown by RT-PCR using primers spanning N-terminal (exons 1-4) and C-terminal (exons 9-11) regions of the gene (Fig. 1D).Fig. 1.


Scaffold attachment factor B2 (SAFB2)- mice reveal non-redundant functions of SAFB2 compared with its paralog, SAFB1.

Jiang S, Katz TA, Garee JP, DeMayo FJ, Lee AV, Oesterreich S - Dis Model Mech (2015)

Generation of SAFB2−/− mice. (A) SAFB2 mouse allele, and targeting construct for deletion of the SAFB2 genomic fragment from exons 4 to 10. SB, Southern blot probe. (B) Southern blot analysis of genomic DNA from embryonic stem (ES) cells (left panel) and mice (right panel). SAFB2+/+ and SAFB2−/− bands have sizes of 12 kb and 5 kb, respectively. (C) Northern blot analysis of RNA from SAFB2+/+, SAFB2+/− and SAFB2−/− mice. (D) RT-PCR using RNA from SAFB2+/+, SAFB2+/− and SAFB2−/− mice and primers targeting both the N and C terminal regions of SAFB2 and SAFB1. NTC, non-template control. (E) Immunoblot of lysates from either SAFB2+/+ or SAFB2−/− mice with antibodies targeting SAFB2 and SAFB1. Lower panel shows an entire SAFB2 immunoblot to provide proof of expression of the N-terminal fragment. (F) LacZ (β-galactosidase) staining of SAFB2+/+ or SAFB2−/− Sertoli cells. Blue, DAPI; red, Alexa Fluor 546 (lacZ).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582101&req=5

DMM019885F1: Generation of SAFB2−/− mice. (A) SAFB2 mouse allele, and targeting construct for deletion of the SAFB2 genomic fragment from exons 4 to 10. SB, Southern blot probe. (B) Southern blot analysis of genomic DNA from embryonic stem (ES) cells (left panel) and mice (right panel). SAFB2+/+ and SAFB2−/− bands have sizes of 12 kb and 5 kb, respectively. (C) Northern blot analysis of RNA from SAFB2+/+, SAFB2+/− and SAFB2−/− mice. (D) RT-PCR using RNA from SAFB2+/+, SAFB2+/− and SAFB2−/− mice and primers targeting both the N and C terminal regions of SAFB2 and SAFB1. NTC, non-template control. (E) Immunoblot of lysates from either SAFB2+/+ or SAFB2−/− mice with antibodies targeting SAFB2 and SAFB1. Lower panel shows an entire SAFB2 immunoblot to provide proof of expression of the N-terminal fragment. (F) LacZ (β-galactosidase) staining of SAFB2+/+ or SAFB2−/− Sertoli cells. Blue, DAPI; red, Alexa Fluor 546 (lacZ).
Mentions: SAFB2−/− mice were generated by homologous recombination in 129/Sv ES cells, replacing exons 4-10 with an IRES-lacZ-SV40pA (β-galactosidase) marker and a PGK-Neomycin-bGHpA selection marker (Fig. 1A). The targeting construct was linearized and electroporated into ES cells, and the neomycin-resistant ES cell colonies were screened by Southern blot analysis (Fig. 1B, left panel). Three recombinant clones were injected into C57BL/6 blastocysts, and two (B11 and G12) were transmitted to germ line. The chimeric mice were bred with wild-type C57BL/6 mice to produce F1 mixed background (129/Sv and C57BL/6) heterozygous mutant mice (SAFB2+/−). Heterozygous F1 mice from each line were intercrossed to produce two independent lines of homozygous mutant mice (SAFB2−/−). Genotyping was confirmed by Southern blot analysis (Fig. 1B, right panel). Loss of SAFB2 mRNA expression was confirmed by northern blot analysis (Fig. 1C), and by RT-PCR assays using primers spanning the Safb2 C-terminus (exons 18-21) (Fig. 1D). RT-PCR with primers covering exons 4-7 revealed remaining expression of the N-terminus. SAFB1 mRNA expression was not affected, as shown by RT-PCR using primers spanning N-terminal (exons 1-4) and C-terminal (exons 9-11) regions of the gene (Fig. 1D).Fig. 1.

Bottom Line: Scaffold attachment factors SAFB1 and SAFB2 are multifunctional proteins that share >70% sequence similarity.Further analysis showed a significantly increased testis weight in SAFB2(-/-) mice, which was associated with an increased number of Sertoli cells.Our data suggest that this is at least in part caused by alterations in androgen-receptor function and expression upon deletion of SAFB2.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Lester and Sue Smith Breast Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

No MeSH data available.


Related in: MedlinePlus