title: Complete reduction of p53 expression by RNA interference following heterozygous knockout in porcine fibroblasts.
abstract: 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.
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, 119 Dandae-ro, Cheonan, Chungnam, 31116, South Korea.|Institute of Green Bioscience and Technology, Seoul National University, Pyeongchang, 25354, South Korea.
authors.forename: Young June
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Institute of Tissue Regeneration Engineering, Dankook University, Cheonan, 31116, South Korea.
authors.forename: Tae-Hyun
authors.initials: TH
authors.lastname: Kim
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, 119 Dandae-ro, Cheonan, Chungnam, 31116, South Korea.
authors.forename: Minjeong
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, 119 Dandae-ro, Cheonan, Chungnam, 31116, South Korea.
authors.forename: Min Ju
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, 119 Dandae-ro, Cheonan, Chungnam, 31116, South Korea.|Institute of Tissue Regeneration Engineering, Dankook University, Cheonan, 31116, South Korea.
authors.forename: Hae-Won
authors.initials: HW
authors.lastname: Kim
authors.affiliation: Department of Nanobiomedical Science and BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, 119 Dandae-ro, Cheonan, Chungnam, 31116, South Korea. shim@dku.edu.|Institute of Tissue Regeneration Engineering, Dankook University, Cheonan, 31116, South Korea. shim@dku.edu.|Department of Physiology, School of Medicine, Dankook University, Cheonan, 31116, South Korea. shim@dku.edu.
authors.forename: Hosup
authors.initials: H
authors.lastname: Shim
mesh_terms: D000818:Animals; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D006579:Heterozygote; D006801:Humans; D051379:Mice; D009154:Mutation; D034622:RNA Interference; D013552:Swine; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D016159:Tumor Suppressor Protein p53
keywords: Knockdown; Knockout; Pig; p53