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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

Confirmation of the processing and knockdown effect of Srsf3 from pMA14 in mouse cell lines. (A) NIH3T3 mouse fibroblast and 69 mouse breast cancer cells were transfected with pBS/U6-loxP (Cre/loxP recombined form of the pBS/U6-ploxPneo plasmid 10), re-pJR22, re-pMA13 and re-pMA14, which are post-recombination forms of pBS/U6-ploxPneo, pJR22, pMA13 and pMA14. 48 h after the transfection, 40 μg of total RNAs were analyzed by Northern blot with a denaturing 15% polyacrylamide gel. Pre-shRNAs and guide-strand siRNAs were detected by pooled 32P-labeled probes complementary to guide-strand siRNA sequences of pJR22, pMA13 and pMA14. Identities of each band are indicated on the right. (B-D) Confirmation of the knockdown effect of Srsf3 shRNA from re-pMA14 in mouse cell lines. NK, NIH3T3 and 69 mouse cells are transfected twice with pBS/U6-loxP or re-pMA14 plasmid for 96 h with an interval of 48 h between transfections, and analyzed by Western blot for protein expression (B), real-time RT-PCR for mRNA expression (C), and WST-8 cell proliferation assay (D). Relative intensity of Srsf3 signal (%) is indicated in (B), with the expression level from pBS/U6-loxP vector-transfected cells set to 100%. Mean ± SD is shown in (C) and (D). *, p<0.05; **, p<0.01 by Student's t-test (n = 3).
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Figure 2: Confirmation of the processing and knockdown effect of Srsf3 from pMA14 in mouse cell lines. (A) NIH3T3 mouse fibroblast and 69 mouse breast cancer cells were transfected with pBS/U6-loxP (Cre/loxP recombined form of the pBS/U6-ploxPneo plasmid 10), re-pJR22, re-pMA13 and re-pMA14, which are post-recombination forms of pBS/U6-ploxPneo, pJR22, pMA13 and pMA14. 48 h after the transfection, 40 μg of total RNAs were analyzed by Northern blot with a denaturing 15% polyacrylamide gel. Pre-shRNAs and guide-strand siRNAs were detected by pooled 32P-labeled probes complementary to guide-strand siRNA sequences of pJR22, pMA13 and pMA14. Identities of each band are indicated on the right. (B-D) Confirmation of the knockdown effect of Srsf3 shRNA from re-pMA14 in mouse cell lines. NK, NIH3T3 and 69 mouse cells are transfected twice with pBS/U6-loxP or re-pMA14 plasmid for 96 h with an interval of 48 h between transfections, and analyzed by Western blot for protein expression (B), real-time RT-PCR for mRNA expression (C), and WST-8 cell proliferation assay (D). Relative intensity of Srsf3 signal (%) is indicated in (B), with the expression level from pBS/U6-loxP vector-transfected cells set to 100%. Mean ± SD is shown in (C) and (D). *, p<0.05; **, p<0.01 by Student's t-test (n = 3).

Mentions: In order to avoid an embryonic lethality owing to an ubiquitous knockout of Srsf3 14, we attempted to knockdown Srsf3 in a tissue-specific manner by Cre/loxP system. In this strategy, we apply a conditional shRNA expression system with a U6 promoter, divided into two segments by floxed neor (neomycin-resistant gene) sequence (Fig. 1A) 10, 15, 16. In this system, ~2 kb of neor insertion disrupts U6 promoter activity in the absence of Cre recombinase. Then, Cre/loxP recombination removes neor to activate U6 promoter and initiate Srsf3 shRNA expression (Fig. 1A). To select an optimal shRNA for Srsf3 knockdown, we constructed three shRNA expression plasmids, pJR22, pMA13 and pMA14, by inserting Srsf3 shRNAs into pBS/U6-ploxPneo plasmid (Fig. 1B)10 to target a splice junction (pJR22 and pMA13) or exon region (pMA14) of Srsf3 (Fig. 1C) and compared them for their processing efficiencies into siRNAs in mouse cells. In order to achieve shRNA expression in mouse cell lines that do not express Cre recombinase, pJR22, pMA13 and pMA14 were processed for Cre/loxP recombination by transformation of Cre-expressing E. coli BNN 132 strain. The resulting post-recombination forms of individual plasmids purified from the BNN 132 strain were renamed as re-pJR22, re-pMA13 and re-pMA14. Then, we transfected re-pJR22, re-pMA13 and re-pMA14 into NIH3T3 mouse fibroblast and 69 mouse breast cancer cells, with pBS/U6-loxP, a Cre/loxP recombination form of pBS/U6-ploxPneo plasmid 10, as a negative transfection control. Forty-eight h after the transfection, total RNAs were analyzed by Northern blotting for precursor shRNA expression and production of guide-strand siRNA (Fig. 2A). Notably, we found an efficient production of the guide-strand siRNA from re-pMA14 (Fig. 2A, lanes 6 and 11), but not from re-pJR22 (Fig. 2A, lanes 4 and 9) or re-pMA13 (Fig. 2A, lanes 5 and 10). Given an efficient processing of pMA14-derived shRNA, we further examined knockdown effect of Srsf3 following transfection of re-pMA14 in NIH3T3, NK and 69 cells. As a result, we found re-pMA14 transfection consistently suppresses Srsf3 expression at protein (Fig. 2B) and mRNA (Fig. 2C) levels over the pBS/U6-loxP transfected cells. In addition, we also found a significant retardation of cell growth after the re-pMA14 transfection of NK or NIH3T3 cells when compared with the pBS/U6-loxP transfected cells (Fig. 2D), consisting with the previous observations that Srsf3 plays an essential role in cell proliferation 8, 17.


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)

Confirmation of the processing and knockdown effect of Srsf3 from pMA14 in mouse cell lines. (A) NIH3T3 mouse fibroblast and 69 mouse breast cancer cells were transfected with pBS/U6-loxP (Cre/loxP recombined form of the pBS/U6-ploxPneo plasmid 10), re-pJR22, re-pMA13 and re-pMA14, which are post-recombination forms of pBS/U6-ploxPneo, pJR22, pMA13 and pMA14. 48 h after the transfection, 40 μg of total RNAs were analyzed by Northern blot with a denaturing 15% polyacrylamide gel. Pre-shRNAs and guide-strand siRNAs were detected by pooled 32P-labeled probes complementary to guide-strand siRNA sequences of pJR22, pMA13 and pMA14. Identities of each band are indicated on the right. (B-D) Confirmation of the knockdown effect of Srsf3 shRNA from re-pMA14 in mouse cell lines. NK, NIH3T3 and 69 mouse cells are transfected twice with pBS/U6-loxP or re-pMA14 plasmid for 96 h with an interval of 48 h between transfections, and analyzed by Western blot for protein expression (B), real-time RT-PCR for mRNA expression (C), and WST-8 cell proliferation assay (D). Relative intensity of Srsf3 signal (%) is indicated in (B), with the expression level from pBS/U6-loxP vector-transfected cells set to 100%. Mean ± SD is shown in (C) and (D). *, p<0.05; **, p<0.01 by Student's t-test (n = 3).
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Figure 2: Confirmation of the processing and knockdown effect of Srsf3 from pMA14 in mouse cell lines. (A) NIH3T3 mouse fibroblast and 69 mouse breast cancer cells were transfected with pBS/U6-loxP (Cre/loxP recombined form of the pBS/U6-ploxPneo plasmid 10), re-pJR22, re-pMA13 and re-pMA14, which are post-recombination forms of pBS/U6-ploxPneo, pJR22, pMA13 and pMA14. 48 h after the transfection, 40 μg of total RNAs were analyzed by Northern blot with a denaturing 15% polyacrylamide gel. Pre-shRNAs and guide-strand siRNAs were detected by pooled 32P-labeled probes complementary to guide-strand siRNA sequences of pJR22, pMA13 and pMA14. Identities of each band are indicated on the right. (B-D) Confirmation of the knockdown effect of Srsf3 shRNA from re-pMA14 in mouse cell lines. NK, NIH3T3 and 69 mouse cells are transfected twice with pBS/U6-loxP or re-pMA14 plasmid for 96 h with an interval of 48 h between transfections, and analyzed by Western blot for protein expression (B), real-time RT-PCR for mRNA expression (C), and WST-8 cell proliferation assay (D). Relative intensity of Srsf3 signal (%) is indicated in (B), with the expression level from pBS/U6-loxP vector-transfected cells set to 100%. Mean ± SD is shown in (C) and (D). *, p<0.05; **, p<0.01 by Student's t-test (n = 3).
Mentions: In order to avoid an embryonic lethality owing to an ubiquitous knockout of Srsf3 14, we attempted to knockdown Srsf3 in a tissue-specific manner by Cre/loxP system. In this strategy, we apply a conditional shRNA expression system with a U6 promoter, divided into two segments by floxed neor (neomycin-resistant gene) sequence (Fig. 1A) 10, 15, 16. In this system, ~2 kb of neor insertion disrupts U6 promoter activity in the absence of Cre recombinase. Then, Cre/loxP recombination removes neor to activate U6 promoter and initiate Srsf3 shRNA expression (Fig. 1A). To select an optimal shRNA for Srsf3 knockdown, we constructed three shRNA expression plasmids, pJR22, pMA13 and pMA14, by inserting Srsf3 shRNAs into pBS/U6-ploxPneo plasmid (Fig. 1B)10 to target a splice junction (pJR22 and pMA13) or exon region (pMA14) of Srsf3 (Fig. 1C) and compared them for their processing efficiencies into siRNAs in mouse cells. In order to achieve shRNA expression in mouse cell lines that do not express Cre recombinase, pJR22, pMA13 and pMA14 were processed for Cre/loxP recombination by transformation of Cre-expressing E. coli BNN 132 strain. The resulting post-recombination forms of individual plasmids purified from the BNN 132 strain were renamed as re-pJR22, re-pMA13 and re-pMA14. Then, we transfected re-pJR22, re-pMA13 and re-pMA14 into NIH3T3 mouse fibroblast and 69 mouse breast cancer cells, with pBS/U6-loxP, a Cre/loxP recombination form of pBS/U6-ploxPneo plasmid 10, as a negative transfection control. Forty-eight h after the transfection, total RNAs were analyzed by Northern blotting for precursor shRNA expression and production of guide-strand siRNA (Fig. 2A). Notably, we found an efficient production of the guide-strand siRNA from re-pMA14 (Fig. 2A, lanes 6 and 11), but not from re-pJR22 (Fig. 2A, lanes 4 and 9) or re-pMA13 (Fig. 2A, lanes 5 and 10). Given an efficient processing of pMA14-derived shRNA, we further examined knockdown effect of Srsf3 following transfection of re-pMA14 in NIH3T3, NK and 69 cells. As a result, we found re-pMA14 transfection consistently suppresses Srsf3 expression at protein (Fig. 2B) and mRNA (Fig. 2C) levels over the pBS/U6-loxP transfected cells. In addition, we also found a significant retardation of cell growth after the re-pMA14 transfection of NK or NIH3T3 cells when compared with the pBS/U6-loxP transfected cells (Fig. 2D), consisting with the previous observations that Srsf3 plays an essential role in cell proliferation 8, 17.

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