Method for inducing reprogramming of skin fibroblast and application thereof

A fibroblast and reprogramming technology, applied in the field of cells, can solve the problems of low reprogramming efficiency and poor stability of iPS cells

Inactive Publication Date: 2018-08-10
深圳市瑞亚再生医学科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In summary, when traditional methods are used to induce reprogramming of skin fibroblasts, the reprogramming efficiency is low, and the stability of the iPS cells produced is poor

Method used

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  • Method for inducing reprogramming of skin fibroblast and application thereof
  • Method for inducing reprogramming of skin fibroblast and application thereof
  • Method for inducing reprogramming of skin fibroblast and application thereof

Examples

Experimental program
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preparation example Construction

[0064] In the method for preparing iPS cells described above, transcription factors and ASF1A-FD genes are introduced into skin fibroblasts. Wherein the transcription factors include Oct3 / 4, Sox2, Klf4 and c-Myc, and the ASF1A-FD gene includes the gene encoding the 1st amino acid to the 154th amino acid of the ASF1A protein. The experimental results show that the introduction of ASF1A-FD gene and OSKM four genes into skin fibroblasts can greatly improve the reprogramming efficiency of human iPS cells, and its reprogramming efficiency is 13.8 times that of the traditional OSKM four gene introduction method, and the obtained iPS The cells can maintain pluripotency for a long time in in vitro culture and passage, which solves the defects of low reprogramming efficiency and easy differentiation during the passage of traditional iPS reprogramming methods.

[0065] In summary, the above method for preparing iPS cells, introducing transcription factors and ASF1A-FD genes into skin fi...

Embodiment 1

[0100] Construction of Retroviral Expression Vector pMXs-ASF1A-FD Containing ASF1A Functional Domain

[0101] 1. Selection of ASF1A functional domain

[0102] For the functional domain of ASF1A protein selected in this example, refer to GenBank: CAG33628.1 amino acid No. 1 to amino acid No. 154, labeled as ASF1A-FD.

[0103] 2. Construction of retroviral expression vector containing ASF1A-FD

[0104] The gene encoding ASF1A-FD is artificially synthesized, and the nucleotide sequence of the ASF1A-FD gene is shown in SEQ ID No.1. A BamH I restriction enzyme site CGGATCCG (shown in SEQ ID No.2) is added at the 5' end, and an XhoI restriction enzyme site CCTCGAGG (shown in SEQ ID No.3) is added at the 3' end. The gene fragment synthesized above was cloned into the retroviral vector pMXs (Cell Biolabs, RTV-010) by BamH I and XhoI double digestion, and the constructed retroviral expression vector pMXs-ASF1A-FD was double digested by BamH I and XhoI. Enzyme digestion for identific...

Embodiment 2

[0106] Preparation of retrovirus

[0107] Select PLAT-A cells as retrovirus packaging cells, which are obtained from human embryonic kidney HEK293 cells by stably transfecting gag, pol and env genes. HEK293 cells transfected with gag, pol and env genes can tolerate a single plasmid Transfection enables rapid production of retroviruses. ASF1A-FD retroviruses and Oct3 / 4, Sox2, Klf4, and c-MYC retroviruses were packaged and prepared using this system, and Oct3 / 4, Sox2, Klf4, and c-MYC retrovirus expression plasmids were all purchased from Addgene (Cat. No. 17217, 17218, 17219, 17220 respectively). The ASF1A-FD retrovirus expression plasmid was prepared according to the method in Example 1.

[0108] The detailed experimental steps of retrovirus preparation are as follows:

[0109] (1) Day1: Inoculate Plat-A cells into a 10cm culture dish, 5×10 6 pcs / dish, 6 dishes in total, at 37°C, 5% CO 2 Incubate overnight in an incubator.

[0110] (2) Day2: 2 hours before plasmid transfe...

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Abstract

The invention discloses a method for inducing reprogramming of skin fibroblast and an application thereof. Transcription factors and an ASF1A-FD genes are introduced into the skin fibroblast, whereinthe transcription factors include Oct3/4, Sox2, Klf4 and c-Myc. The ASF1A-FD genes include genes for coding No.1 amino acid-No.154 amino acid of ASF1A protein. By introducing= the ASF1A-FD genes and the OSKM transcription factors (Oct3/4, Sox2, Klf4 and c-Myc) into the skin fibroblast, reprogramming efficiency can be greatly improved.

Description

technical field [0001] The invention relates to the field of cells, in particular to a method for inducing skin fibroblast reprogramming and its application. Background technique [0002] Embryonic Stem Cells (ESCs) have attractive application prospects in drug screening, regenerative medicine, and research on disease pathogenesis. However, due to limitations in material sources, ethical pressure, and operating techniques, the development of ESCs is extremely slow. Four exogenous transcription factors Oct3 / 4, Sox2, Klf4, and c-Myc (OSKM) are introduced into somatic cells through viral vectors, and induced pluripotent stem cells (induced pluripotent stem cells, iPS cells) with similar characteristics to ESCs can be induced. This method not only avoids the ethical disputes involved in the acquisition of ESCs in the past, but also is simple to operate and has a rich source of materials. It is expected to obtain individual-specific treatment materials according to the needs of p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N5/077
CPCC12N5/0653C12N5/0655C12N5/0657C12N5/0696C12N15/86C12N2500/38C12N2500/40C12N2501/065C12N2501/15C12N2501/33C12N2501/39C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2501/999C12N2506/45C12N2510/00C12N2730/00043C12N2800/107
Inventor 不公告发明人
Owner 深圳市瑞亚再生医学科技有限责任公司
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