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Adapted Resistance to the Knockdown Effect of shRNA-Derived Srsf3 siRNAs in Mouse Littermates.

Ajiro M, Jia R, Wang RH, Deng CX, Zheng ZM - Int. J. Biol. Sci. (2015)

Bottom Line: Gene silencing techniques are widely used to control gene expression and have potential for RNAi-based therapeutics.Although a small portion of the transgenic mouse littermates were found to produce siRNAs in the targeted tissues, most of the transgenic littermates at two months of age failed to display a knockdown phenotype of Srsf3 expression in their liver and mammary gland tissues where an abundant level of Srsf3 siRNAs remained.Data indicate that the host resistance to a gene-specific siRNA targeting an essential gene transcript can be developed in animals, presumably as a physiological necessity to cope with the hostile perturbation.

View Article: PubMed Central - PubMed

Affiliation: 1. Tumor Virus RNA Biology Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA.

ABSTRACT
Gene silencing techniques are widely used to control gene expression and have potential for RNAi-based therapeutics. In this report, transgenic mouse lines were created for conditional knockdown of Srsf3 (SRp20) expression in liver and mammary gland tissues by expressing Srsf3-specific shRNAs driven by a U6 promoter. Although a small portion of the transgenic mouse littermates were found to produce siRNAs in the targeted tissues, most of the transgenic littermates at two months of age failed to display a knockdown phenotype of Srsf3 expression in their liver and mammary gland tissues where an abundant level of Srsf3 siRNAs remained. We saw only one of four mice with liver/mammary gland expressing Srsf3 siRNA displayed a suppressed level of Srsf3 protein, but not the mRNA. Data indicate that the host resistance to a gene-specific siRNA targeting an essential gene transcript can be developed in animals, presumably as a physiological necessity to cope with the hostile perturbation.

No MeSH data available.


Related in: MedlinePlus

Identification of the founder mice for conditional Srsf3 shRNA expression and confirmation of the tissue-specific recombination. (A) Primers (F1, F2 and R1) used for the founder screening and Cre/loxP recombination. (B) PCR screening for 47 mice derived from oocytes with a pronuclear injection of linearized pMA-14. Mouse genomic DNAs from tail snips were analyzed by PCR with a primer set of F1 and R1. Three (#34, #37 and #40) out of 47 mice were identified as the founder mice, which we named as shRNA/34, shRNA/37 and shRNA/40. P, plasmid pMA14 positive control. (C) shRNA/37 and shRNA/40 were bred with Alb-Cre or MMTV-Cre mice, and the resulting Alb-Cre:shRNA/40 (male), MMTV-Cre:shRNA/40 (female), and MMTV-Cre:shRNA/37 (female) were analyzed for the Cre/loxP recombination in individual tissues at 2 months of age. PCR was performed for genomic DNAs extracted from individual tissues with primer sets of F2+R1 to detect a post-recombination form and F1+R1 to detect a pre-recombination form of pBS/U6-ploxPneo-derived pMA14 plasmid, respectively. (D) Summary of reproductive function and Cre-mediated recombination of the founder mice of shRNA/34, shRNA/37 and shRNA/40.
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Figure 3: Identification of the founder mice for conditional Srsf3 shRNA expression and confirmation of the tissue-specific recombination. (A) Primers (F1, F2 and R1) used for the founder screening and Cre/loxP recombination. (B) PCR screening for 47 mice derived from oocytes with a pronuclear injection of linearized pMA-14. Mouse genomic DNAs from tail snips were analyzed by PCR with a primer set of F1 and R1. Three (#34, #37 and #40) out of 47 mice were identified as the founder mice, which we named as shRNA/34, shRNA/37 and shRNA/40. P, plasmid pMA14 positive control. (C) shRNA/37 and shRNA/40 were bred with Alb-Cre or MMTV-Cre mice, and the resulting Alb-Cre:shRNA/40 (male), MMTV-Cre:shRNA/40 (female), and MMTV-Cre:shRNA/37 (female) were analyzed for the Cre/loxP recombination in individual tissues at 2 months of age. PCR was performed for genomic DNAs extracted from individual tissues with primer sets of F2+R1 to detect a post-recombination form and F1+R1 to detect a pre-recombination form of pBS/U6-ploxPneo-derived pMA14 plasmid, respectively. (D) Summary of reproductive function and Cre-mediated recombination of the founder mice of shRNA/34, shRNA/37 and shRNA/40.

Mentions: After confirming the function of shRNA derived from pMA14 in mouse cells, we next generated the transgenic mice for its conditional expression. We conducted a pronuclear injection of linearized pMA14 plasmid into FVB/N mouse oocytes, and the founder mice were screened by genotyping PCR with a primer set of F1 and R1 for genomic DNA extracted from tail snips (Fig. 3A). As a result, from 47 mice derived from the injected oocytes, we identified three founder female mice, which we named shRNA/34, shRNA/37 and shRNA/40 (Fig. 3B). In addition, we found the offspring of shRNA/37 founder mouse do not express shRNA even in the presence of Cre recombinase, as discussed later, and only shRNA/40 strain showed both reproductive function and conditional shRNA expression.


Adapted Resistance to the Knockdown Effect of shRNA-Derived Srsf3 siRNAs in Mouse Littermates.

Ajiro M, Jia R, Wang RH, Deng CX, Zheng ZM - Int. J. Biol. Sci. (2015)

Identification of the founder mice for conditional Srsf3 shRNA expression and confirmation of the tissue-specific recombination. (A) Primers (F1, F2 and R1) used for the founder screening and Cre/loxP recombination. (B) PCR screening for 47 mice derived from oocytes with a pronuclear injection of linearized pMA-14. Mouse genomic DNAs from tail snips were analyzed by PCR with a primer set of F1 and R1. Three (#34, #37 and #40) out of 47 mice were identified as the founder mice, which we named as shRNA/34, shRNA/37 and shRNA/40. P, plasmid pMA14 positive control. (C) shRNA/37 and shRNA/40 were bred with Alb-Cre or MMTV-Cre mice, and the resulting Alb-Cre:shRNA/40 (male), MMTV-Cre:shRNA/40 (female), and MMTV-Cre:shRNA/37 (female) were analyzed for the Cre/loxP recombination in individual tissues at 2 months of age. PCR was performed for genomic DNAs extracted from individual tissues with primer sets of F2+R1 to detect a post-recombination form and F1+R1 to detect a pre-recombination form of pBS/U6-ploxPneo-derived pMA14 plasmid, respectively. (D) Summary of reproductive function and Cre-mediated recombination of the founder mice of shRNA/34, shRNA/37 and shRNA/40.
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Figure 3: Identification of the founder mice for conditional Srsf3 shRNA expression and confirmation of the tissue-specific recombination. (A) Primers (F1, F2 and R1) used for the founder screening and Cre/loxP recombination. (B) PCR screening for 47 mice derived from oocytes with a pronuclear injection of linearized pMA-14. Mouse genomic DNAs from tail snips were analyzed by PCR with a primer set of F1 and R1. Three (#34, #37 and #40) out of 47 mice were identified as the founder mice, which we named as shRNA/34, shRNA/37 and shRNA/40. P, plasmid pMA14 positive control. (C) shRNA/37 and shRNA/40 were bred with Alb-Cre or MMTV-Cre mice, and the resulting Alb-Cre:shRNA/40 (male), MMTV-Cre:shRNA/40 (female), and MMTV-Cre:shRNA/37 (female) were analyzed for the Cre/loxP recombination in individual tissues at 2 months of age. PCR was performed for genomic DNAs extracted from individual tissues with primer sets of F2+R1 to detect a post-recombination form and F1+R1 to detect a pre-recombination form of pBS/U6-ploxPneo-derived pMA14 plasmid, respectively. (D) Summary of reproductive function and Cre-mediated recombination of the founder mice of shRNA/34, shRNA/37 and shRNA/40.
Mentions: After confirming the function of shRNA derived from pMA14 in mouse cells, we next generated the transgenic mice for its conditional expression. We conducted a pronuclear injection of linearized pMA14 plasmid into FVB/N mouse oocytes, and the founder mice were screened by genotyping PCR with a primer set of F1 and R1 for genomic DNA extracted from tail snips (Fig. 3A). As a result, from 47 mice derived from the injected oocytes, we identified three founder female mice, which we named shRNA/34, shRNA/37 and shRNA/40 (Fig. 3B). In addition, we found the offspring of shRNA/37 founder mouse do not express shRNA even in the presence of Cre recombinase, as discussed later, and only shRNA/40 strain showed both reproductive function and conditional shRNA expression.

Bottom Line: Gene silencing techniques are widely used to control gene expression and have potential for RNAi-based therapeutics.Although a small portion of the transgenic mouse littermates were found to produce siRNAs in the targeted tissues, most of the transgenic littermates at two months of age failed to display a knockdown phenotype of Srsf3 expression in their liver and mammary gland tissues where an abundant level of Srsf3 siRNAs remained.Data indicate that the host resistance to a gene-specific siRNA targeting an essential gene transcript can be developed in animals, presumably as a physiological necessity to cope with the hostile perturbation.

View Article: PubMed Central - PubMed

Affiliation: 1. Tumor Virus RNA Biology Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA.

ABSTRACT
Gene silencing techniques are widely used to control gene expression and have potential for RNAi-based therapeutics. In this report, transgenic mouse lines were created for conditional knockdown of Srsf3 (SRp20) expression in liver and mammary gland tissues by expressing Srsf3-specific shRNAs driven by a U6 promoter. Although a small portion of the transgenic mouse littermates were found to produce siRNAs in the targeted tissues, most of the transgenic littermates at two months of age failed to display a knockdown phenotype of Srsf3 expression in their liver and mammary gland tissues where an abundant level of Srsf3 siRNAs remained. We saw only one of four mice with liver/mammary gland expressing Srsf3 siRNA displayed a suppressed level of Srsf3 protein, but not the mRNA. Data indicate that the host resistance to a gene-specific siRNA targeting an essential gene transcript can be developed in animals, presumably as a physiological necessity to cope with the hostile perturbation.

No MeSH data available.


Related in: MedlinePlus