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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 shRNA expression system targeting Srsf3. (A) A diagram of the conditional shRNA expression system. In this system, U6 promoter is inactivated by the insertion of ~2 kb of floxed neor sequence. Cre/loxP recombination remove the neor sequence and reactivates U6 promoter to initiate transcription of Srsf3 shRNA. SPH, SphI post-octamer homology; OCT, octamer motif; PSE, proximal sequence element; TATA, TATA box. (B) Construction of the conditional shRNA expression plasmids, pJR22, pMA13 and pMA14. Srsf3 shRNA was inserted downstream of the U6-neor in pBS/U6-ploxPneo10. Guide- and passenger-strand sequences are indicated by open boxes. Nucleotide positions of Srsf3 target sites of each shRNA are indicated according to Mus musculus Srsf3 mRNA (GenBank: NM_013663.5). The maturation process of transcribed shRNA is illustrated below. (C) Diagram of shRNA target sites on mouse Srsf3 mRNA and comparison with human SRSF3 mRNA sequence (GenBank: NM_003017.4). Plasmid pJR22 and pMA13 are designed to express a shRNA targeting the exon 2 and 3 junction, and pMA14 for targeting the exon 2 of Srsf3. Nucleotides not conserved in human SRSF3 mRNA are underlined.
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Figure 1: Conditional shRNA expression system targeting Srsf3. (A) A diagram of the conditional shRNA expression system. In this system, U6 promoter is inactivated by the insertion of ~2 kb of floxed neor sequence. Cre/loxP recombination remove the neor sequence and reactivates U6 promoter to initiate transcription of Srsf3 shRNA. SPH, SphI post-octamer homology; OCT, octamer motif; PSE, proximal sequence element; TATA, TATA box. (B) Construction of the conditional shRNA expression plasmids, pJR22, pMA13 and pMA14. Srsf3 shRNA was inserted downstream of the U6-neor in pBS/U6-ploxPneo10. Guide- and passenger-strand sequences are indicated by open boxes. Nucleotide positions of Srsf3 target sites of each shRNA are indicated according to Mus musculus Srsf3 mRNA (GenBank: NM_013663.5). The maturation process of transcribed shRNA is illustrated below. (C) Diagram of shRNA target sites on mouse Srsf3 mRNA and comparison with human SRSF3 mRNA sequence (GenBank: NM_003017.4). Plasmid pJR22 and pMA13 are designed to express a shRNA targeting the exon 2 and 3 junction, and pMA14 for targeting the exon 2 of Srsf3. Nucleotides not conserved in human SRSF3 mRNA are underlined.

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)

Conditional shRNA expression system targeting Srsf3. (A) A diagram of the conditional shRNA expression system. In this system, U6 promoter is inactivated by the insertion of ~2 kb of floxed neor sequence. Cre/loxP recombination remove the neor sequence and reactivates U6 promoter to initiate transcription of Srsf3 shRNA. SPH, SphI post-octamer homology; OCT, octamer motif; PSE, proximal sequence element; TATA, TATA box. (B) Construction of the conditional shRNA expression plasmids, pJR22, pMA13 and pMA14. Srsf3 shRNA was inserted downstream of the U6-neor in pBS/U6-ploxPneo10. Guide- and passenger-strand sequences are indicated by open boxes. Nucleotide positions of Srsf3 target sites of each shRNA are indicated according to Mus musculus Srsf3 mRNA (GenBank: NM_013663.5). The maturation process of transcribed shRNA is illustrated below. (C) Diagram of shRNA target sites on mouse Srsf3 mRNA and comparison with human SRSF3 mRNA sequence (GenBank: NM_003017.4). Plasmid pJR22 and pMA13 are designed to express a shRNA targeting the exon 2 and 3 junction, and pMA14 for targeting the exon 2 of Srsf3. Nucleotides not conserved in human SRSF3 mRNA are underlined.
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Related In: Results  -  Collection

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Figure 1: Conditional shRNA expression system targeting Srsf3. (A) A diagram of the conditional shRNA expression system. In this system, U6 promoter is inactivated by the insertion of ~2 kb of floxed neor sequence. Cre/loxP recombination remove the neor sequence and reactivates U6 promoter to initiate transcription of Srsf3 shRNA. SPH, SphI post-octamer homology; OCT, octamer motif; PSE, proximal sequence element; TATA, TATA box. (B) Construction of the conditional shRNA expression plasmids, pJR22, pMA13 and pMA14. Srsf3 shRNA was inserted downstream of the U6-neor in pBS/U6-ploxPneo10. Guide- and passenger-strand sequences are indicated by open boxes. Nucleotide positions of Srsf3 target sites of each shRNA are indicated according to Mus musculus Srsf3 mRNA (GenBank: NM_013663.5). The maturation process of transcribed shRNA is illustrated below. (C) Diagram of shRNA target sites on mouse Srsf3 mRNA and comparison with human SRSF3 mRNA sequence (GenBank: NM_003017.4). Plasmid pJR22 and pMA13 are designed to express a shRNA targeting the exon 2 and 3 junction, and pMA14 for targeting the exon 2 of Srsf3. Nucleotides not conserved in human SRSF3 mRNA are underlined.
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.