CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs

Wu, Jun; Vilariño, M.; Suzuki, Keiichiro; Okamura, Daiji; Bogliotti, Y. S.; Park, In Sung; Rowe, Joan D; McNabb, Bret R.; Ross, Pablo J.; Belmonte, Juan Carlos Izpisúa

doi:10.1038/s41598-017-08596-5

Cited by 41 publications

(34 citation statements)

References 15 publications

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“…(), who reported that supplementation with 50 μmol/L CGA is advantageous to the porcine IVP system. When porcine zygotes from oocytes matured with or without 50 μmol/L CGA were electroporated with Cas9 messenger RNA and single guide RNA targeting sites in pancreatic duodenal homeobox‐1 ( Pdx1 ) gene (Wu et al., ), the proportion (89.3%, 25/28) of blastocysts with a mutated sequence in the CGA‐treated group was similar with that (88.2%, 30/34) in the untreated group (data not shown). These observations indicate that CGA supplementation during maturation culture may increase the number of embryos with insertions or deletions (indels) in the targeted gene by electropolation.…”

Section: Discussionmentioning

confidence: 91%

Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization

Nguyen

Wittayarat

Kim

et al. 2018

Animal Science Journal

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Electroporation is the technique of choice to introduce an exogenous gene into embryos for transgenic animal production. Although this technique is practical and effective, embryonic damage caused by electroporation treatment remains a major problem. This study was conducted to evaluate the optimal culture system for electroporation-treated porcine embryos by supplementation of chlorogenic acid (CGA), a potent antioxidant, during in vitro oocyte maturation. The oocytes were treated with various concentrations of CGA (0, 10, 50, and 100 μmol/L) through the duration of maturation for 44 hr. The treated oocytes were then fertilized, electroporated at 30 V/mm with five 1 msec unipolar pulses, and subsequently cultured in vitro until development into the blastocyst stage. Without electroporation, the treatment with 50 μmol/L CGA had useful effects on the maturation rate of oocytes, the total cell number, and the apoptotic nucleus indices of blastocysts. When the oocytes were electroporated after in vitro fertilization, the treatment with 50 μmol/L CGA supplementation significantly improved the rate of oocytes that developed into blastocysts and reduced the apoptotic nucleus indices (4.7% and 7.6, respectively) compared with those of the untreated group (1.4% and 13.0, respectively). These results suggested that supplementation with 50 μmol/L CGA during maturation improves porcine embryonic development and quality of electroporation-treated embryos.

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Section: Discussionmentioning

confidence: 91%

Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization

Nguyen

Wittayarat

Kim

et al. 2018

Animal Science Journal

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“…Pancreatic duodenal homeobox 1 ( PDX1 , also known as IPF‐1 , IDX‐1 , and STF‐1 ) is a crucial gene for pancreas development during the fetal period (Bonal & Herrera, 2008; Jonsson, Carlsson, Edlund, & Edlund, 1994; McKinnon & Docherty, 2001). The biallelic mutation of PDX1 leads to agenesis of the pancreas both in mice (Jonsson et al, 1994) and in pigs (Kang et al, 2017; Wu, Vilarino et al, 2017), and results in infant death, whereas in mice, monoallelic disruption impairs insulin secretion from pancreatic β‐cells (Brissova et al, 2002). To our knowledge, however, viable PDX1 monoallelic mutants have not been investigated in pigs because PDX1 disrupted pigs were generated by somatic cell nuclear transfer (SCNT) with PDX1 biallelic mutant cells in the previous study (Kang et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Generation of viable PDX1 gene‐edited founder pigs as providers of nonmosaics

Tanihara

Hirata

Nguyen

et al. 2020

Molecular Reproduction Devel

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Pancreatic duodenal homeobox 1 (PDX1) is a crucial gene for pancreas development during the fetal period. PDX1-modified pigs have the potential to be used as a model of diabetes mellitus. However, the severe health problems caused by the PDX1 mutation limit phenotypic studies of PDX1-modified pigs as diabetes models. In this study, we generated PDX1-modified pigs by the CRISPR/Cas9 system introduced into zygotes via electroporation and investigated the mosaicism, phenotypes, and inheritance of the resulting pigs. After the embryo transfer of PDX1-modified zygotes, nine mutant piglets were delivered. Two piglets were apancreatic biallelic mutants. For the other seven piglets, the ratio of mutant alleles to total alleles was 17.5-79.7%. Two mutant piglets with high mutation rates (67.7% and 79.7%) exhibited hypoplasia of the pancreas, whereas the other five piglets were healthy.One of the male mutant piglets was further analyzed. The ejaculated semen from the pig contained PDX1-mutant spermatozoa and the pig showed normal reproductive ability. In conclusion, the frequency of the PDX1 mutation is presumed to relate to pancreas formation, and PDX1 mutant founder pigs generated from zygotes introduced to the CRISPR/Cas9 system can serve as providers of nonmosaics to contribute to medical research on diabetes mellitus. K E Y W O R D SCRISPR/Cas9, diabetes mellitus, electroporation, IVF, PDX1-mutant pigs SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section.

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“…In brief, precise genome editing technologies are widely used in cattle and other domestic animals to improve transgenesis (Menchaca et al, 2016;Niu et al, 2017;Wu et al, 2017), and most of these studies rely on TN technology to produce live offspring. It is possible that the knowledge gained through the CRISPR-Cas 9 system may soon be used in cloning production.…”

Section: Perspectives In Animal Transgenesis and Conclusionmentioning

confidence: 99%

Achievements and perspectives in cloned and transgenic cattle production by nuclear transfer: influence of cell type, epigenetic status and new technology

Curcio¹,

Bressan²,

Meirelles³

et al. 2017

Anim Reprod

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Genetically modified cattle production is motivated by many factors, including recombinant protein production for therapeutic purposes, disease models and animals presenting improved production traits. Nuclear transfer (NT), combined with efficient cultivation methods, genetic modification and donor cell selection is important for transgenic cattle production. Studies have found that adult cells (such as fibroblasts and cumulus cells, among others) used as nuclear donors achieved results similar to those of fetal cells, with the advantages of easier collection and a known genotype/phenotype. However, no consensus has been reached on the influence of cell type on transgene expression levels and post-reprogramming capacity after nuclear transfer, and these factors appear to be related to epigenetic factors. The development of new strategies, such as chromatin-modifying agents (CMAs), in vitro generation of induced pluripotent cells (iPS cells) and precise genome editing techniques are being explored and may influence nuclear reprogramming success for efficiently producing genetically modified bovine clones.

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CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs

Cited by 41 publications

References 15 publications

Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization

Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization

Generation of viable PDX1 gene‐edited founder pigs as providers of nonmosaics

Achievements and perspectives in cloned and transgenic cattle production by nuclear transfer: influence of cell type, epigenetic status and new technology

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