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Method for simultaneously activating transcriptional activity of multiple porcine endogenous stem cell factors by adopting tandem sgRNAs

A stem cell factor, endogenous technology, applied in the direction of retroRNA virus, animal cells, vertebrate cells, etc., can solve the problems that the expression of stem cell factor is not effectively activated, and the efficiency of iPSC is low

Active Publication Date: 2019-09-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficiency of pig iPSCs is currently low, and exogenous overexpression of OSKM and miR-302 / 367 can induce iPSCs, but the expression of endogenous stem cell factors has not been effectively activated

Method used

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  • Method for simultaneously activating transcriptional activity of multiple porcine endogenous stem cell factors by adopting tandem sgRNAs
  • Method for simultaneously activating transcriptional activity of multiple porcine endogenous stem cell factors by adopting tandem sgRNAs
  • Method for simultaneously activating transcriptional activity of multiple porcine endogenous stem cell factors by adopting tandem sgRNAs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Construction and detection of PK-15 cell lines stably expressing dCAS-VP64 and MS2-P65-HSF1

[0026] In order to stably express dCAS-VP64 and MS2-P65-HSF1 in PK-15 cells, they were introduced by lentivirus. Pack lenti dCAS-VP64_Blast lentivirus, inoculate appropriate amount of HEK 293T cells in a 10cm culture dish 24 hours before lentivirus packaging, at 37°C, 5% CO 2 Cultured in an incubator, the induction medium is DMEM containing 10% fetal bovine serum. When the cell density reached 80% the next day, dilute 30 μL lipofectamine2000 with 470 μL opti-MEM, and dilute 24 μg plasmid DNA with opti-MEM medium, including PMD2. , #61425) 12μg, the three were mixed and allowed to stand for 5 minutes, and added to HEK293T cells in a 10cm dish that had been changed. Replace with fresh DMEM induction culture medium 6 hours after transfection; recover virus supernatants at 32 hours and 60 hours after transfection, treat the supernatants with a 0.45 μm filter membrane, ...

Embodiment 2

[0037] Example 2. Application of PK-15-dCas9-MS2 stable cell line to screen sgRNA targeting porcine endogenous Oct4, Sox2, Klf4 and c-Myc, miR-302 / 367 promoter activity

[0038] 2.1 Construction and sequencing verification of porcine Oct4, Sox2, Klf4 and c-Myc, miR-302 / 367 promoter dual luciferase reporter gene vectors

[0039] In order to quickly assess the activities of different sgRNAs targeting and activating different stem cell factor promoters, certain length promoter regions of Oct4, Sox2, Klf4, c-Myc, and miR-302 / 367 were respectively amplified by PCR (primers are listed in Table 5). Linked to the dual-luciferase vector pGL3-Basic, five dual-luciferase reporter gene expression plasmids were constructed, and identified by sequencing and restriction enzyme digestion, and named pGL3-Oct4, pGL3-Sox2, pGL3- Klf4, pGL3-c-Myc.

[0040] Table 5 PCR primers of OSKM, miR-302 / 367 promoter region

[0041]

[0042] Note: The underlined Xho I site: CTCGAG, HindIII cleavage site...

Embodiment 3

[0057] Example 3. The use of PK-15-dCas9-MS2 stable cell line and tandem sgRNA expression vector can realize the synchronous activation of transcription of porcine endogenous Oct4, Sox2, Klf4, c-Myc and miR-302 / 367

[0058] In order to test whether the CRISPR / dCas9 activation strategy can simultaneously promote the expression of OSKM and miR-302 / 367 in cells, sg6_Oct4, sg4_Sox2, sg4_Klf4, sg1_c-Myc and sg4_miR-302 / 367 were connected in series (see sequence 1 for the sequence), and The sequence was synthesized by Nanjing GenScript. Ligate the tandem sequences in the lenti-sgRNA(MS2)-zeo linear vector. And verified by Nhe1 and BamH1 double enzyme digestion experiments (such as Figure 10 ). Subsequently, PK-dCas9-MS cells were transfected with overexpression tandem sgRNA and dual fluorescein promoter activity detection vectors, and it was found that the activity of sgRNA targeting the Oct4 gene promoter was enhanced by about 1.6 times (such as Figure 11 -A), the sgRNA activi...

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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.

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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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
CPCC07K14/47C12N5/0686C12N15/86C12N2510/02C12N2740/15043
Inventor 谢胜松周玲刘敏苗义良刘海龙徐德全
Owner HUAZHONG AGRI UNIV
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