Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts
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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.
KeywordsKnockout Knockdown p53 Pig
This research was supported by a grant (PJ011375) from the Next-Generation BioGreen 21 Program, Rural Development Administration, a grant (2014034046) supported by the Bio & Medical Technology Development Program, and a grant (2009–0093829) supported by the Priority Research Centers Program through National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning.
- Ahn KS, Kim YJ, Kim M, Lee BH, Heo SY, Kang MJ, Kang YK, Lee JW, Lee KK, Kim JH, Nho WG, Hwang SS, Woo JS, Park JK, Park SB, Shim H (2011) Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts. Theriogenology 75:933–939CrossRefPubMedGoogle Scholar
- Basel MT, Balivada S, Beck AP, Kerrigan MA, Pyle MM, Dekkers JC, Wyatt CR, Rowland RR, Anderson DE, Bossmann SH, Troyer DL (2012) Human xenografts are not rejected in a naturally occurring immunodeficient porcine line: a human tumor model in pigs. Biores Open Access 1:63–68CrossRefPubMedPubMedCentralGoogle Scholar
- Fontemaggi G, Dell’Orso S, Trisciuoglio D, Shay T, Melucci E, Fazi F, Terrenato I, Mottolese M, Muti P, Domany E, Del Bufalo D, Strano S, Blandino G (2009) The execution of the transcriptional axis mutant p53, E2F1 and ID4 promotes tumor neo-angiogenesis. Nat Struct Mol Biol 16:1086–1093CrossRefPubMedGoogle Scholar
- Leuchs S, Saalfrank A, Merkl C, Flisikowska T, Edlinger M, Durkovic M, Rezaei N, Kurome M, Zakhartchenko V, Kessler B, Flisikowski K, Kind A, Wolf E, Schnieke A (2012) Inactivation and inducible oncogenic mutation of p53 in gene targeted pigs. PLoS One 7:e43323CrossRefPubMedPubMedCentralGoogle Scholar
- Sieren JC, Meyerholz DK, Wang XJ, Davis BT, Newell JD Jr, Hammond E, Rohret JA, Rohret FA, Struzynski JT, Goeken JA, Naumann PW, Leidinger MR, Taghiyev A, Van Rheeden R, Hagen J, Darbro BW, Quelle DE, Rogers CS (2014) Development and translational imaging of a TP53 porcine tumorigenesis model. J Clin Invest 124:4052–4066CrossRefPubMedPubMedCentralGoogle Scholar
- Tovar C, Graves B, Packman K, Filipovic Z, Higgins B, Xia M, Tardell C, Garrido R, Lee E, Kolinsky K, To KH, Linn M, Podlaski F, Wovkulich P, Vu B, Vassilev LT (2013) MDM2 small-molecule antagonist RG7112 activates p53 signaling and regresses human tumors in preclinical cancer models. Cancer Res 73:2587–2597CrossRefPubMedGoogle Scholar