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Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer

Overview of attention for article published in Journal of Translational Medicine, November 2017
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Title
Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer
Published in
Journal of Translational Medicine, November 2017
DOI 10.1186/s12967-017-1327-0
Pubmed ID
Authors

Youfeng Shen, Kaixiang Xu, Zaimei Yuan, Jianxiong Guo, Heng Zhao, Xuezeng Zhang, Lu Zhao, Yubo Qing, Honghui Li, Weirong Pan, Baoyu Jia, Hong-Ye Zhao, Hong-Jiang Wei

Abstract

Pigs have many features that make them attractive as biomedical models for various diseases, including cancer. P53 is an important tumor suppressor gene that exerts a central role in protecting cells from oncogenic transformation and is mutated in a large number of human cancers. P53 mutations occur in almost every type of tumor and in over 50% of all tumors. In a recent publication, pigs with a mutated P53 gene were generated that resulted in lymphoma and renal and osteogenic tumors. However, approximately 80% of human tumors have dysfunctional P53. A P53-deficient pig model is still required to elucidate. Transcription activator-like effector nucleases (TALENs) were designed to target porcine P53 exon 4. The targeting activity was evaluated using a luciferase SSA recombination assay. P53 biallelic knockout (KO) cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs followed by electroporation with TALENs plasmids. One cell line was selected as the donor cell line for somatic cell nuclear transfer (SCNT) for the generation of P53 KO pigs. P53 KO stillborn fetuses and living piglets were obtained. Gene typing of the collected cloned individuals was performed by T7EI assay and sequencing. Fibroblast cells from Diannan miniature piglets with a P53 biallelic knockout or wild type were analyzed for the P53 response to doxorubicin treatment by confocal microscopy and western blotting. The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 55.35-fold higher than those of the control. Eight cell lines (8/19) were mutated for P53, and five of them were biallelic knockouts. One of the biallelic knockout cell lines was selected as nuclear donor cells for SCNT. The cloned embryos were transferred into five recipient gilts, three of them becoming pregnant. Five live fetuses were obtained from one surrogate by caesarean section after 38 days of gestation for genotyping. Finally, six live piglets and one stillborn piglet were collected from two recipients by caesarean section. Sequencing analyses of the target site confirmed the P53 biallelic knockout in all fetuses and piglets, consistent with the genotype of the donor cells. The qPCR analysis showed that the expression of the P53 mRNA had significant reduction in various tissues of the knockout piglets. Furthermore, confocal microscopy and western blotting analyses demonstrated that the fibroblast cells of Diannan miniature piglets with a P53 biallelic knockout were defective in mediating DNA damage when incubated with doxorubicin. TALENs combined with SCNT was successfully used to generate P53 KO Diannan miniature pigs. Although these genetically engineered Diannan miniature pigs had no tumorigenic signs, the P53 gene was dysfunctional. We believe that these pigs will provide powerful new resources for preclinical oncology and basic cancer research.

Twitter Demographics

The data shown below were collected from the profiles of 3 tweeters who shared this research output. Click here to find out more about how the information was compiled.

Mendeley readers

The data shown below were compiled from readership statistics for 10 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 10 100%

Demographic breakdown

Readers by professional status Count As %
Researcher 2 20%
Student > Doctoral Student 2 20%
Student > Bachelor 2 20%
Student > Ph. D. Student 1 10%
Librarian 1 10%
Other 0 0%
Unknown 2 20%
Readers by discipline Count As %
Agricultural and Biological Sciences 4 40%
Arts and Humanities 1 10%
Biochemistry, Genetics and Molecular Biology 1 10%
Social Sciences 1 10%
Chemistry 1 10%
Other 0 0%
Unknown 2 20%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 05 November 2017.
All research outputs
#10,657,436
of 14,011,199 outputs
Outputs from Journal of Translational Medicine
#2,118
of 2,719 outputs
Outputs of similar age
#214,561
of 317,910 outputs
Outputs of similar age from Journal of Translational Medicine
#250
of 261 outputs
Altmetric has tracked 14,011,199 research outputs across all sources so far. This one is in the 20th percentile – i.e., 20% of other outputs scored the same or lower than it.
So far Altmetric has tracked 2,719 research outputs from this source. They typically receive a little more attention than average, with a mean Attention Score of 7.2. This one is in the 18th percentile – i.e., 18% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 317,910 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 26th percentile – i.e., 26% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 261 others from the same source and published within six weeks on either side of this one. This one is in the 2nd percentile – i.e., 2% of its contemporaries scored the same or lower than it.