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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.


Conditional expression of Srsf3 shRNAs in transgenic mice and their knockdown efficiency in liver tissues. (A-C) Tissue-specific expression of Srsf3 shRNAs in transgenic mice. Northern blot was conducted for 40 μg of total RNAs extracted from individual tissues from Alb-Cre:shRNA/40 (male) (A, lanes 2-4 and C, lanes 2-7), Alb-Cre:shRNA/37 (male) (B, lanes 2-9), MMTV-Cre:shRNA/37 (female) (A, lanes 5-8) and MMTV-Cre:shRNA/40 (female) (A, lanes 9-12), at 2 months of age. The shRNA and its precursor were detected by a 32P-labeled probe complementary to the guide-strand shRNA from pMA14. Eight μg of total RNA from re-pMA14-transfected NIH3T3 cells served as a positive control (A, lane 13, and B, lane 10). Total RNA was separated by a denaturing 15% polyacrylamide gel. (D-F) Alb-Cre:shRNA/40 (n=3) and Alb-Cre (n=2) male littermates were analyzed for their body weight (D) and liver weight (E) at 2 months of age. Representative pictures of livers from 2-month-old Alb-Cre:shRNA/40 and Alb-Cre male littermates are shown in (F). Scale bar, 1 cm. (G-I) Liver tissues of two-month-old littermates of Alb-Cre:shRNA/40 and Alb-Cre (a, b, c, d, e, f, and g indicate individual mice) were analyzed by Northern blotting for Srsf3 shRNA expression (G), by real-time RT-PCR for Srsf3 mRNA expression (H), and by Western blotting for protein expression (I). Relative intensity (%) of Srsf3 protein signal is indicated in (I), with Srsf3 protein level in Alb-Cre control liver set to 100%, after normalizing with Gapdh. (J) Mammary gland tissues of two-month-old MMTV-Cre:shRNA/40 and adult MMTV-Cre female mice are analyzed by Western blotting for Srsf3 protein expression after normalizing with Gapdh which served as a control for sample loading, with Srsf3 level in MMTV-Cre mammary gland tissues set to 100%. HCT116 cell lysate served as a positive control in (I). Mean±SD is shown in (D), (E) and (H). NS, p>0.05 by Student's t-test.
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Figure 4: Conditional expression of Srsf3 shRNAs in transgenic mice and their knockdown efficiency in liver tissues. (A-C) Tissue-specific expression of Srsf3 shRNAs in transgenic mice. Northern blot was conducted for 40 μg of total RNAs extracted from individual tissues from Alb-Cre:shRNA/40 (male) (A, lanes 2-4 and C, lanes 2-7), Alb-Cre:shRNA/37 (male) (B, lanes 2-9), MMTV-Cre:shRNA/37 (female) (A, lanes 5-8) and MMTV-Cre:shRNA/40 (female) (A, lanes 9-12), at 2 months of age. The shRNA and its precursor were detected by a 32P-labeled probe complementary to the guide-strand shRNA from pMA14. Eight μg of total RNA from re-pMA14-transfected NIH3T3 cells served as a positive control (A, lane 13, and B, lane 10). Total RNA was separated by a denaturing 15% polyacrylamide gel. (D-F) Alb-Cre:shRNA/40 (n=3) and Alb-Cre (n=2) male littermates were analyzed for their body weight (D) and liver weight (E) at 2 months of age. Representative pictures of livers from 2-month-old Alb-Cre:shRNA/40 and Alb-Cre male littermates are shown in (F). Scale bar, 1 cm. (G-I) Liver tissues of two-month-old littermates of Alb-Cre:shRNA/40 and Alb-Cre (a, b, c, d, e, f, and g indicate individual mice) were analyzed by Northern blotting for Srsf3 shRNA expression (G), by real-time RT-PCR for Srsf3 mRNA expression (H), and by Western blotting for protein expression (I). Relative intensity (%) of Srsf3 protein signal is indicated in (I), with Srsf3 protein level in Alb-Cre control liver set to 100%, after normalizing with Gapdh. (J) Mammary gland tissues of two-month-old MMTV-Cre:shRNA/40 and adult MMTV-Cre female mice are analyzed by Western blotting for Srsf3 protein expression after normalizing with Gapdh which served as a control for sample loading, with Srsf3 level in MMTV-Cre mammary gland tissues set to 100%. HCT116 cell lysate served as a positive control in (I). Mean±SD is shown in (D), (E) and (H). NS, p>0.05 by Student's t-test.

Mentions: To confirm tissue-specific Srsf3 shRNA expression in Alb-Cre:shRNA/40, Alb-Cre:shRNA/37, MMTV-Cre:shRNA/37, and MMTV-Cre:shRNA/40 mice, total RNA was extracted from individual tissues from 2-month-old mice and Srsf3 shRNA expression was evaluated by Northern blotting with a probe complementary to the guide-strand siRNA from pMA14. In consistent with the presence of efficient Cre/loxP recombination (Fig. 3C), Alb-Cre:shRNA/40 showed Srsf3 siRNA production primarily in liver and weakly in salivary gland, but not in kidney, lung, heart and spleen tissues (Fig. 4A, lanes 2-4, Fig. 4C, lanes 2-7). MMTV-Cre:shRNA/40 showed Srsf3 siRNA expression in salivary gland, kidney, liver and mammary gland (Fig. 4A, lanes 9-12). In contrast, MMTV-Cre:shRNA/37 and Alb-Cre:shRNA/37 did not show Srsf3 siRNA expression in all tissues examined (Fig. 4A, lanes 5-8, and Fig. 4B, lanes 2-9) in spite of successful Cre/loxP recombination in these tissues in MMTV-Cre:shRNA/37 (Fig. 3C) and Alb-Cre:shRNA/37 was positive in genotyping for Alb-Cre. These observations suggest that the shRNA expression construct had integrated into a chromosomal locus which was accessible by Cre recombinase, but was transcriptionally inactive in shRNA/37 strain. Thus, the offsprings of shRNA/37 founder mouse did not express shRNAs even in the presence of Cre recombinase. In the subsequent studies, only the shRNA/40 strain was used for further studies. We investigated the liver-specific Srsf3 shRNA expression in Alb-Cre:shRNA/40 mice (Fig. 4C). In Alb-Cre:shRNA/40 and Alb-Cre male littermates, there was no significant difference in body weight (Fig. 4D), liver weight (Fig. 4E), and liver morphology (Fig. 4F) at 2 months of age. By comparing individual Alb-Cre:shRNA/40 littermates for their expression levels of Srsf3 shRNA, Srsf3 mRNA, and Srsf3 protein in liver tissues (Fig. 4G-I), we confirmed the production of Srsf3-specific siRNAs in Alb-Cre:shRNA/40 littermates (Fig. 4G, lanes 2, 5 and 7), but not in Alb-Cre littermates (Fig. 4G, lanes 3, 4, 6 and 8). However, Srsf3 shRNA expression was found not always being accompanied with a reduced level of Srsf3 protein in these liver tissues.


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)

Conditional expression of Srsf3 shRNAs in transgenic mice and their knockdown efficiency in liver tissues. (A-C) Tissue-specific expression of Srsf3 shRNAs in transgenic mice. Northern blot was conducted for 40 μg of total RNAs extracted from individual tissues from Alb-Cre:shRNA/40 (male) (A, lanes 2-4 and C, lanes 2-7), Alb-Cre:shRNA/37 (male) (B, lanes 2-9), MMTV-Cre:shRNA/37 (female) (A, lanes 5-8) and MMTV-Cre:shRNA/40 (female) (A, lanes 9-12), at 2 months of age. The shRNA and its precursor were detected by a 32P-labeled probe complementary to the guide-strand shRNA from pMA14. Eight μg of total RNA from re-pMA14-transfected NIH3T3 cells served as a positive control (A, lane 13, and B, lane 10). Total RNA was separated by a denaturing 15% polyacrylamide gel. (D-F) Alb-Cre:shRNA/40 (n=3) and Alb-Cre (n=2) male littermates were analyzed for their body weight (D) and liver weight (E) at 2 months of age. Representative pictures of livers from 2-month-old Alb-Cre:shRNA/40 and Alb-Cre male littermates are shown in (F). Scale bar, 1 cm. (G-I) Liver tissues of two-month-old littermates of Alb-Cre:shRNA/40 and Alb-Cre (a, b, c, d, e, f, and g indicate individual mice) were analyzed by Northern blotting for Srsf3 shRNA expression (G), by real-time RT-PCR for Srsf3 mRNA expression (H), and by Western blotting for protein expression (I). Relative intensity (%) of Srsf3 protein signal is indicated in (I), with Srsf3 protein level in Alb-Cre control liver set to 100%, after normalizing with Gapdh. (J) Mammary gland tissues of two-month-old MMTV-Cre:shRNA/40 and adult MMTV-Cre female mice are analyzed by Western blotting for Srsf3 protein expression after normalizing with Gapdh which served as a control for sample loading, with Srsf3 level in MMTV-Cre mammary gland tissues set to 100%. HCT116 cell lysate served as a positive control in (I). Mean±SD is shown in (D), (E) and (H). NS, p>0.05 by Student's t-test.
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Figure 4: Conditional expression of Srsf3 shRNAs in transgenic mice and their knockdown efficiency in liver tissues. (A-C) Tissue-specific expression of Srsf3 shRNAs in transgenic mice. Northern blot was conducted for 40 μg of total RNAs extracted from individual tissues from Alb-Cre:shRNA/40 (male) (A, lanes 2-4 and C, lanes 2-7), Alb-Cre:shRNA/37 (male) (B, lanes 2-9), MMTV-Cre:shRNA/37 (female) (A, lanes 5-8) and MMTV-Cre:shRNA/40 (female) (A, lanes 9-12), at 2 months of age. The shRNA and its precursor were detected by a 32P-labeled probe complementary to the guide-strand shRNA from pMA14. Eight μg of total RNA from re-pMA14-transfected NIH3T3 cells served as a positive control (A, lane 13, and B, lane 10). Total RNA was separated by a denaturing 15% polyacrylamide gel. (D-F) Alb-Cre:shRNA/40 (n=3) and Alb-Cre (n=2) male littermates were analyzed for their body weight (D) and liver weight (E) at 2 months of age. Representative pictures of livers from 2-month-old Alb-Cre:shRNA/40 and Alb-Cre male littermates are shown in (F). Scale bar, 1 cm. (G-I) Liver tissues of two-month-old littermates of Alb-Cre:shRNA/40 and Alb-Cre (a, b, c, d, e, f, and g indicate individual mice) were analyzed by Northern blotting for Srsf3 shRNA expression (G), by real-time RT-PCR for Srsf3 mRNA expression (H), and by Western blotting for protein expression (I). Relative intensity (%) of Srsf3 protein signal is indicated in (I), with Srsf3 protein level in Alb-Cre control liver set to 100%, after normalizing with Gapdh. (J) Mammary gland tissues of two-month-old MMTV-Cre:shRNA/40 and adult MMTV-Cre female mice are analyzed by Western blotting for Srsf3 protein expression after normalizing with Gapdh which served as a control for sample loading, with Srsf3 level in MMTV-Cre mammary gland tissues set to 100%. HCT116 cell lysate served as a positive control in (I). Mean±SD is shown in (D), (E) and (H). NS, p>0.05 by Student's t-test.
Mentions: To confirm tissue-specific Srsf3 shRNA expression in Alb-Cre:shRNA/40, Alb-Cre:shRNA/37, MMTV-Cre:shRNA/37, and MMTV-Cre:shRNA/40 mice, total RNA was extracted from individual tissues from 2-month-old mice and Srsf3 shRNA expression was evaluated by Northern blotting with a probe complementary to the guide-strand siRNA from pMA14. In consistent with the presence of efficient Cre/loxP recombination (Fig. 3C), Alb-Cre:shRNA/40 showed Srsf3 siRNA production primarily in liver and weakly in salivary gland, but not in kidney, lung, heart and spleen tissues (Fig. 4A, lanes 2-4, Fig. 4C, lanes 2-7). MMTV-Cre:shRNA/40 showed Srsf3 siRNA expression in salivary gland, kidney, liver and mammary gland (Fig. 4A, lanes 9-12). In contrast, MMTV-Cre:shRNA/37 and Alb-Cre:shRNA/37 did not show Srsf3 siRNA expression in all tissues examined (Fig. 4A, lanes 5-8, and Fig. 4B, lanes 2-9) in spite of successful Cre/loxP recombination in these tissues in MMTV-Cre:shRNA/37 (Fig. 3C) and Alb-Cre:shRNA/37 was positive in genotyping for Alb-Cre. These observations suggest that the shRNA expression construct had integrated into a chromosomal locus which was accessible by Cre recombinase, but was transcriptionally inactive in shRNA/37 strain. Thus, the offsprings of shRNA/37 founder mouse did not express shRNAs even in the presence of Cre recombinase. In the subsequent studies, only the shRNA/40 strain was used for further studies. We investigated the liver-specific Srsf3 shRNA expression in Alb-Cre:shRNA/40 mice (Fig. 4C). In Alb-Cre:shRNA/40 and Alb-Cre male littermates, there was no significant difference in body weight (Fig. 4D), liver weight (Fig. 4E), and liver morphology (Fig. 4F) at 2 months of age. By comparing individual Alb-Cre:shRNA/40 littermates for their expression levels of Srsf3 shRNA, Srsf3 mRNA, and Srsf3 protein in liver tissues (Fig. 4G-I), we confirmed the production of Srsf3-specific siRNAs in Alb-Cre:shRNA/40 littermates (Fig. 4G, lanes 2, 5 and 7), but not in Alb-Cre littermates (Fig. 4G, lanes 3, 4, 6 and 8). However, Srsf3 shRNA expression was found not always being accompanied with a reduced level of Srsf3 protein in these liver tissues.

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.