Method of using annulet free carrier to efficiently reprogramming blood cells to generate iPSC

A technology of dissociated vectors and blood cells, which is applied in the field of efficient reprogramming of blood cells to generate iPSCs with microcircular dissociated vectors, which can solve problems such as increased safety hazards, abnormal karyotype, and low reprogramming efficiency, and achieve high transfection efficiency Effect

Active Publication Date: 2018-08-17
安徽中盛溯源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Since the viral vectors used to transduce foreign genes into donor cells will have a certain impact on them during the reprogramming process, there are gene rearrangements, karyotype abnormalities, epigenetic abnormalities, etc. in the clones screened by this method phenomenon, even with a high risk of carcinogenicity
In 2008, Okita et al. obtained mouse iPSCs through multiple conventional plasmid transfection methods, but the operation was cumbersome and the reprogramming efficiency was low
[0022] In 2010, Jia et al. used the Minicircle vector to obtain non-integrated iPSCs from human adipose-derived mesenchymal stem cells, but multiple transfections were required, the operation was cumbersome, and the efficiency was limited.
However, partially reprogrammed iPSCs cannot silence the expression of exogenous viral transgenes, which increases the risk of tumorigenicity and greatly limits its clinical application.

Method used

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  • Method of using annulet free carrier to efficiently reprogramming blood cells to generate iPSC
  • Method of using annulet free carrier to efficiently reprogramming blood cells to generate iPSC
  • Method of using annulet free carrier to efficiently reprogramming blood cells to generate iPSC

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 The construction idea of ​​minicircle DNA parental plasmid

[0071] The microcircle DNA parental plasmid construction strategy of the present invention is shown in the figure figure 1 shown. The attB in the microcircle DNA parent plasmid in the figure is the bacterial attachment site of recombinase Ф31; attP is the phage attachment site; 32×Sce-I sites contain 32 consecutive I-SceI restriction enzyme sites; pUC ORI plasmid replication origin; Kan R kanamycin resistance gene; SV40poly-A is the PolyA tailing signal of Simian vacuolar virus; Transgene indicates the nucleotide sequence of the inserted foreign gene; P is the promoter of the foreign gene expression cassette , generally a conventional promoter, preferably a eukaryotic promoter, more particularly a pEF1α promoter or a CMV promoter. The process of site-specific recombination of the microcircle DNA parent plasmid in the host bacteria is as follows: the microcircle DNA parent plasmid has attB sites and...

Embodiment 2

[0075] Example 2 Construction of pMEP4-E-AxOct4, pMEP4-ESCLM2L and pMEP4-ENET minicircle DNA parental plasmids

[0076] Construct minicircle DNA parental plasmids via the following protocol

[0077] S1. Synthesize an empty minicircle DNA plasmid containing attB, attP, and 30×I-SceI restriction endonuclease cutting site sequences through a commercial company.

[0078] S2, respectively for the attached figure 2 The shown pEP4-E-AxOct4, pEP4-ESCLM2L and pEP4-ENET free-type vectors were double-digested, and the results of the digestion were identified by agarose gel electrophoresis, and nucleotide fragments of different sizes were obtained after digestion.

[0079] S3. Use T4 DNA ligase to insert the obtained nucleotide fragments into the empty minicircle DNA plasmid, perform ligation at 4°C overnight or at room temperature for 1 hour, and then add the ligation product to E.coli DH5α strain to sense Transformation was carried out in the state cell suspension, and inoculated on ...

Embodiment 3

[0081] Example 3 Preparation of pMEP4-E-AxOct4, pMEP4-ESCLM2L and pMEP4-ENET minicircle DNA

[0082] Construct minicircle DNA parental plasmids via the following protocol

[0083] S1. Using the pMEP4-E-AxOct4, pMEP4-ESCLM2L and pMEP4-ENET microcircle DNA parental plasmids constructed in Example 2, transform ZYCY10P3S2T engineering bacteria at 37°C, and culture them statically for 14-16 hours;

[0084] S2. Pick a single clone from the engineered bacteria containing the microcircle DNA parental plasmid obtained in S1 and culture it in suspension overnight; add an equal volume of fresh LB medium containing arabinose after overnight, and the final mass volume concentration of arabinose is 0.1%, that is, Contain 1 g of arabinose in 1 L of aqueous solution, incubate for 4 hours at a temperature of 32 °C and a shaking frequency of 250 times / min to induce ZYCY10P3S2T engineering bacteria to express ФC31 integrase and restriction endonuclease I-SceI;

[0085] S3. Contacting the microc...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a method of using annulet free carrier to efficiently reprogramming blood cells to generate iPSC. The method includes following steps: S1, extracting a monocyte from a human blood cell, and selectively culturing the monocyte through an amplification culture medium to obtain a red blood cell ancestral cell; S2, guiding the annulet free carrier expressing exogenous reprogramming factor into the red blood cell ancestral cell obtained in the S1; S3, culturing the red blood cell ancestral cell obtained in the S2 and containing the annulet free carrier expressing the exogenous reprogramming factor through a multipotent stem cell induced culture medium, and inducing in a feed-layer-free system into a reprogramming intermediate-state cell; S4, after complete inducing is finished, replacing the multipotent stem cell induced culture medium in the S3 with a multipotent stem cell culture medium to maintain culturing, and obtaining a cell with expression of the exogenous reprogramming factor expression disappearing and expression of endogenous multipotent genes POU5F1, NANOG, TRA-1-60 and TRA-1-81 activated, namely iPSC.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently reprogramming blood cells to generate iPSCs with a free microcircle carrier. Background technique [0002] In 2006, Takahashi and Yamanaka used retroviral vectors to transfer four transcription factors OSKM (octamer-binding transcription factor 4, Oct4), sex determining region Y box protein 2 (sex determining region Y box protein 2, Sox2), Kuppel-like factor 4 (kruppel-likefactor 4, Klf4) and C-myelocytomatosis oncogene (c-Myc) were transferred into mouse fibroblasts to obtain induced pluripotent stem cells (induced Pluripotent Stem Cell, iPSC). Signed that iPSCs are very similar to embryonic stem cells (ESCs) in terms of biological characteristics, and effectively avoid problems such as ethical restrictions and difficulties in obtaining materials, and have great potential in disease models, drug screening, developmental biology research, and cel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/63C12N15/66
CPCC12N5/0696C12N15/63C12N15/66C12N2501/60C12N2501/602C12N2501/603C12N2501/604C12N2501/605C12N2501/606C12N2501/608C12N2506/11
Inventor 俞君英张健董成友张颖
Owner 安徽中盛溯源生物科技有限公司
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