RNA Interference in Pigs: Comparison of RNAi Test Systems and Expression Vectors

Merkl, Claudia; Leuchs, Simon; Saalfrank, Anja; Kind, Alexander; Schnieke, Angelika

doi:10.1007/s12033-010-9346-6

Cited by 10 publications

(9 citation statements)

References 49 publications

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“…Most of the 10 candidate miRNAs we tested in vitro were effective against ovine and bovine BLG, which recapitulates the successful design of effective RNAi molecules against porcine BLG in a previous study (14). Although a few of our miRNAs showed some target specificity for either of the two tested forms of BLG, this was not correlated with sequence homology between the miRNA and target.…”

Section: E8356 | Wwwpnasorgsupporting

confidence: 82%

“…A recent in vitro study demonstrated the effectiveness of several shRNAs and miRNAs directed against the porcine variant of BLG (14). We used artificial miRNAs based on the murine miRNA-155 to knock down BLG.…”

Section: E8356 | Wwwpnasorgmentioning

confidence: 99%

“…miRNAs can be driven by Pol II promoters, which enable spatiotemporally restricted expression and greatly limit off-target effects that may arise from constitutive miRNA expression. Indeed, constitutive expression of BLG-specific RNAi constructs negatively affects primary cell growth, indicating that abundant interfering RNAs aimed at BLG may be toxic (14). When the same RNAi constructs were controlled by a lactation-specific promoter, they showed no adverse effects and were compatible with early pig development (14).…”

Section: E8356 | Wwwpnasorgmentioning

confidence: 99%

“…Indeed, constitutive expression of BLG-specific RNAi constructs negatively affects primary cell growth, indicating that abundant interfering RNAs aimed at BLG may be toxic (14). When the same RNAi constructs were controlled by a lactation-specific promoter, they showed no adverse effects and were compatible with early pig development (14). However, the feasibility of reducing BLG in milk by an RNAi-mediated approach remains untested.…”

Section: E8356 | Wwwpnasorgmentioning

confidence: 99%

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Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

Jabed

Wagner

McCracken

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Milk from dairy cows contains the protein β-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG. One tandem construct was expressed in the mammary gland of an ovine BLG-expressing mouse model, resulting in 96% knockdown of ovine BLG in milk. Following this in vivo validation, we produced a transgenic calf, engineered to express these tandem miRNAs. Analysis of hormonally induced milk from this calf demonstrated absence of BLG and a concurrent increase of all casein milk proteins. The findings demonstrate miRNA–mediated depletion of an allergenic milk protein in cattle and validate targeted miRNA expression as an effective strategy to alter milk composition and other livestock traits.

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Section: E8356 | Wwwpnasorgsupporting

confidence: 82%

Section: E8356 | Wwwpnasorgmentioning

confidence: 99%

Section: E8356 | Wwwpnasorgmentioning

confidence: 99%

Section: E8356 | Wwwpnasorgmentioning

confidence: 99%

See 2 more Smart Citations

Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

Jabed

Wagner

McCracken

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…40 µg total protein was loaded in each lane and separated by 15% SDS-PAGE. Blotting and detection of porcine p53 and GAPDH was carried out as previously described [33]. Band densities were quantified using the image analysis tool ImageJ (http://rsb.info.nih.gov/ij/).…”

Section: Methodsmentioning

confidence: 99%

Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

et al. 2012

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Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53R167H mutant allele, orthologous to oncogenic human mutant TP53R175H and mouse Trp53R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.

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Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts

Kim

et al. 2016

In Vitro Cell.Dev.Biol.-Animal

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Tumor suppressor p53 plays a critical role in the regulation of cell cycle and apoptosis in mammals. Mutations of p53 often cause various cancers. Murine models have improved our understanding on tumorigenesis associated with p53 mutations. However, mice and humans are different in many ways. For example, the short lifespans of mice limit the clinical application of the data obtained from this species. Porcine model could be an alternative as pigs share many anatomical and physiological similarities with humans. Here, we modified the expression levels of p53 messenger RNA (mRNA) and protein in porcine fetal fibroblasts using a combination of gene targeting and RNA interference. First, we disrupted the p53 gene to produce p53 knockout (KO) cells. Second, the p53 shRNA expression vector was introduced into fibroblasts to isolate p53 knockdown (KD) cells. We obtained p53 KO, KD, and KO + KD fibroblasts which involve p53 KO and KD either separately or simultaneously. The mRNA expression of p53 in p53 KO fibroblasts was similar to that in the wild-type control. However, the mRNA expression levels of p53 in KD and KO + KD cells were significantly decreased. The p53 protein level significant reduced in p53 KD. Interestingly, no p53 protein was detected in KO + KD, suggesting a complete reduction of the protein by synergistic effect of KO and KD. This study demonstrated that various expression levels of p53 in porcine fibroblasts could be achieved by gene targeting and RNA interference. Moreover, complete abolishment of protein expression is feasible using a combination of gene targeting and RNA interference.

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RNA Interference in Pigs: Comparison of RNAi Test Systems and Expression Vectors

Cited by 10 publications

References 49 publications

Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts

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