Molecular Cloning of Pig Rad51, Rad52, and Rad54 Genes, Which Are Involved in Homologous Recombination Machinery

Matsunami, Katsuyoshi; Otsuka, Hideaki; Xu, Hengjie; Firdawes, S.; Yamamoto, Akira; Ishimaru, A.; Fukuzawa, Masahiro; Miyagawa, Shuji

doi:10.1016/j.transproceed.2008.08.023

Cited by 4 publications

(4 citation statements)

References 6 publications

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“…Additionally, previous study showed that RAD51 recombinase was a key player for HR and the repair of DNA DSBs 21 and the cDNA sequence of pig RAD51 gene was successfully isolated by PCR-based methods 22 . In order to investigate RAD51 effects on the indicated pROSA26 site insertion, we constructed pig RAD51-over expression plasmid (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These results indicate that electroporation is a versatile transfection system that may be used to explore the biological functions of mRNAs, siRNAs and various cell lines. For example, siRNAs could be designed to suppress several NHEJ key molecules, including DNA ligase IV, KU70, KU80 and XLF 24, 25 , and mRNAs could be designed that increase the expression of key HDR key molecules, such as RAD51 and RAD52 21, 22, 26 . Then, corresponding siRNAs or mRNAs could be introduced into the PFFs with CRISPR/Cas9 and the donor vector by using this transfection system, which may be used to enhance the site-specific knock-in efficiency.…”

Section: Discussionmentioning

confidence: 99%

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Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Xie

Pang

Wang

et al. 2017

Sci Rep

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Genetically modified pigs have important roles in agriculture and biomedicine. However, genome-specific knock-in techniques in pigs are still in their infancy and optimal strategies have not been extensively investigated. In this study, we performed electroporation to introduce a targeting donor vector (a non-linearized vector that did not contain a promoter or selectable marker) into Porcine Foetal Fibroblasts (PFFs) along with a CRISPR/Cas9 vector. After optimization, the efficiency of the EGFP site-specific knock-in could reach up to 29.6% at the pRosa26 locus in PFFs. Next, we used the EGFP reporter PFFs to address two key conditions in the process of achieving transgenic pigs, the limiting dilution method and the strategy to evaluate the safety and feasibility of the knock-in locus. This study demonstrates that we establish an efficient procedures for the exogenous gene knock-in technique and creates a platform to efficiently generate promoter-less and selectable marker-free transgenic PFFs through the CRISPR/Cas9 system. This study should contribute to the generation of promoter-less and selectable marker-free transgenic pigs and it may provide insights into sophisticated site-specific genome engineering techniques for additional species.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Xie

Pang

Wang

et al. 2017

Sci Rep

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“…Co-Ip proved the existence of Dp71-lamin B1-RAD51 protein complex in HBE. RAD51 is proved to be an essential protein for DNA repair by homologous recombination [14, 15]. Overexpression of Rad51 in different organisms and cell types increased homologous recombination (HR) and increased resistance to DNA damaging agents.…”

Section: Discussionmentioning

confidence: 99%

Dp71 depleted HBE cells displayed increased DNA damage and apoptosis induced by H2O2

et al. 2019

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Human bronchial epithelium (HBE)-Dp71 anti-sense(AS)cells with stably transfected Dp71 siRNA plasmids were prepared for further exploration of Dp71 biological traits in cells other than PC12. HBE-Dp71AS cells displayed increased DNA damage induced by H 2 O 2 . Apoptosis of HBE-Dp71AS cells induced by H 2 O 2 was increased via enhancing caspase 3, caspase 8 and caspase 9. HBE-Dp71AS cells also displayed decreased proliferation and clonogenic formation. RAD51 was proved to be a new binding partner of Dp71 by co-immunoprecipitation (Ip) and immunofluorescence. Reduced RAD51 mRNA and protein levels were observed in HBE-Dp71AS cells. Decreased lamin B1, focal adhesion kinase (FAK), phosphorylated focal adhesion kinase (p-FAK) and phosphorylated protein kinase B (p-AKT) were detected in the HBE-Dp71AS cells, which functioned together with RAD51 as the molecular explanations for the character alterations of HBE-Dp71AS cells. Electronic supplementary material The online version of this article (10.1186/s11658-019-0169-6) contains supplementary material, which is available to authorized users.

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“…Increased frequencies of homologous recombination in stem cell lines ( Tichy et al, 2010 ) is a result of elevated proteins known to be involved in homologous recombination ( Matsunami et al, 2008 ). Stem cells show decreased expression of DNA ligase IV, which is a rate limiting factor for NHEJ ( Riballo et al, 2009 ) in the majority of mammalian cells.…”

Section: Use Of Crispr/cas9 Technology For Genome Editing In Transformentioning

confidence: 99%

Using the CRISPR/Cas9 system to understand neuropeptide biology and regulation

et al. 2017

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Neuropeptides and their receptors play a role in physiological responses such as appetite, stress and inflammatory pain. With neuropeptides having such diverse and important physiological roles, knocking-out the genes encoding them, their receptors, parts of their regulatory sequences, or reproducing disease associated polymorphic variants are important steps in studying neuropeptides and how they may contribute to disease. Previously, knock-outs were generated using methods such as targeted homologous recombination in embryonic stem cells but this method is costly and time-consuming. The CRISPR/Cas9 system has rapidly taken over the genome editing field and will advance our understanding of neuropeptide genes and their regulation. With CRISPR/Cas9 technology, the time and costs involved in producing transgenic animal models, is greatly reduced. In this review, we describe how the system can be used to manipulate genomic sequences by “knock-out” or “knock-in” mutations in cell lines or in animal models. We also discuss the specificity of the system and methods to limit off-target effects. When combined with the availability of genome sequences, CRISPR/Cas9 directed genome editing in vitro and in vivo, promises to provide a deeper understanding of the biology of the neuropeptides in health and disease than has ever been available before.

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Molecular Cloning of Pig Rad51, Rad52, and Rad54 Genes, Which Are Involved in Homologous Recombination Machinery

Cited by 4 publications

References 6 publications

Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Optimization of a CRISPR/Cas9-mediated Knock-in Strategy at the Porcine Rosa26 Locus in Porcine Foetal Fibroblasts

Dp71 depleted HBE cells displayed increased DNA damage and apoptosis induced by H2O2

Using the CRISPR/Cas9 system to understand neuropeptide biology and regulation

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