Synchronous activation of transcriptional activity of multiple porcine endogenous stem cell factors using tandem sgRNAs

A stem cell factor, endogenous technology, applied in retro RNA viruses, animal cells, vertebrate cells, etc., can solve the problems of ineffective activation of stem cell factor expression and low iPSC efficiency.
CN110218741BActive Publication Date: 2020-12-25HUAZHONG AGRI UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
HUAZHONG AGRI UNIV
Publication Date
2020-12-25

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Abstract

The invention provides a method for simultaneously activating multiple porcine endogenous stem cell factors by adopting a tandem sgRNA strategy. Blasticidin and hygromycin drugs are adopted for screening and constructing a PK-15 cell line (PK-15-dCas9-MS2) capable of stably expressing dCAS-VP64 and MS2-P65-HSF1 first, sgRNAs capable of targeting promoters of the porcine stem cell factors Oct4, Sox2, Klf4, c-Myc and miR-302 / 367 are designed and constructed separately, high-activity sgRNA screening is carried out on the cells, the five sgRNAs with the highest gene transcription activation efficiency are serially combined and constructed into an expression vector, and the expression vector is introduced into the PK-15-dCas9-MS2 cells to simultaneously activate the transcriptional activity ofporcine OSKM and miR-302 / 367. The method has a potential application value in induced generation of porcine iPSC.
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Description

technical field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for synchronously activating the transcriptional activity of multiple pig endogenous stem cell factors by adopting a tandem sgRNA strategy. Background technique

[0002] In August 2006, Takahashi and Yamanaka first introduced the four transcription factors of mouse KLF4, Sox2, Oct-3 / 4 and c-Myc (OSKM) into mouse fibroblasts through retrovirus (Mouse embryonic fibroblasts cell MEF cells), MEF cells are reprogrammed to generate induced pluripotent stem cells (iPSC). On this basis, they transferred these four transcription factor vectors into human skin fibroblasts, and successfully induced human iPSCs. Early iPSC induction efficiency was low and the cycle was long. With the continuous development of related technologies, Anokye-Danso et al. found that miR-302 / 367 can effectively increase the number of clones by two orders of magnitude. OSKM and miR-302 / 367 play a...

Claims

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