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Induced pluripotent stem cells, preparation method and application thereof

A pluripotent stem cell and inducible technology, applied in the field of induced pluripotent stem cells and its preparation, can solve problems such as uncontrollable, target gene integration site, insertion copy number cannot be artificially controlled, random transfection, etc.

Active Publication Date: 2021-07-06
HAINAN YILING MEDICAL INDUSTRY & DEVELOPMENT CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason may be that transfection is a relatively random and difficult-to-control process, and the integration site of the target gene and the inserted copy number cannot be controlled by humans.
It is likely that the expression level of each factor introduced must be suitable for the cell to reprogram, and this suitable level may be within a narrow range. Therefore, this random gene introduction results in only a few cells having the ability to reprogram. Conditions for reprogramming
Moreover, when iPS cells are cloned and passaged, the cells are prone to differentiation and apoptosis, and it is difficult to expand and culture
[0004] In summary, the reprogramming efficiency is low when the traditional method is used to prepare iPS cells, and the stability of the iPS cells produced is poor.

Method used

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  • Induced pluripotent stem cells, preparation method and application thereof
  • Induced pluripotent stem cells, preparation method and application thereof
  • Induced pluripotent stem cells, preparation method and application thereof

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preparation example Construction

[0047] A method for preparing induced pluripotent stem cells (iPS) according to one embodiment includes the following steps S110-S120.

[0048]S110. Introducing transcription factors and Erbin gene into skin fibroblasts, wherein the transcription factors include Oct4, Sox2, Klf4 and c-Myc.

[0049] In one embodiment, the skin fibroblasts are human skin fibroblasts. The iPS preparation method of this embodiment can realize the reprogramming from human skin fibroblasts to form human iPS cells, and the reprogramming efficiency is high.

[0050] Among them, the Erbin gene is a new type of gene located on the long arm of human chromosome 5, and the protein expressed by it is referred to as Erbin protein (Erb B2-interacting protein).

[0051] In this embodiment, the sequence of the Erbin gene can be found in NCBI database NM_001253697.1.

[0052] The combination of four transcription factors, Oct4, Sox2, Klf4 and c-Myc, referred to as OSKM, introduces OSKM into somatic cells, whic...

Embodiment 1

[0104] Construction of lentiviral expression vector pSin-EF2-Erbin

[0105] The lentiviral expression plasmid pSin4-EF2-Oct4 (Addgene, 16579) was digested with EcoR I and SpeI, and the bands were separated by agarose gel electrophoresis. The electrophoresis pattern of enzyme digestion products is as follows: figure 1 As shown, there are two clear bands at about 7.5kb and 1.1kb respectively. A 7569bp fragment was recovered by cutting the gel (AxyPrep DNA Gel Recovery Kit), which was the lentiviral expression empty vector pSin-EF2, which was used to construct the lentiviral expression plasmid.

[0106] The gene encoding Erbin (NM_001253697.1) was artificially synthesized, restriction enzyme sites EcoR I and SpeI were added at the 5' end and 3' end respectively, and the gene fragment was cloned into the lentiviral expression empty vector pSin-EF2 to construct The lentiviral expression vector pSin-EF2-Erbin was obtained. The constructed pSin-EF2-Erbin lentiviral expression plas...

Embodiment 2

[0108] Preparation of lentivirus carrying Erbin gene and lentivirus carrying transcription factor

[0109] According to the instruction of Invitrogen Lipofectamine 3000 transfection kit, the pSin-EF2-Erbin plasmid and the lentiviral packaging plasmid PMD2.G (Addgene, 12259) were transfected into 293T cells, cultivated and collected the culture supernatant to obtain the target gene Erbin. Lentivirus (Erbin Lentivirus)

[0110] According to the instruction of Invitrogen Lipofectamine 3000 transfection kit, pSin-EF2-OKSIM (Addgene, 124603) and lentiviral packaging plasmid psPAX2 (Addgene, 12260) were transfected into 293T cells, and the culture supernatant was collected to obtain lentiviral cells carrying OKSIM. Virus (OKSIM lentivirus). Among them, the pSin-EF2-OKSIM plasmid contains the four genes Oct4, Sox2, Klf4 and c-Myc, and the I in the middle represents IRES, which is the internal ribosome entry site sequence (Internal ribosome entry site, IRES), and its function is to i...

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Abstract

The invention discloses an induced pluripotent stem cell and its preparation method and application. Transcription factors and Erbin gene are introduced into skin fibroblasts, and the skin fibroblasts are reprogrammed through cell culture to obtain induced pluripotent stem cells . The experimental results show that introducing Erbin gene and OSKM four genes into skin fibroblasts can improve the reprogramming efficiency of human iPS cells, and the obtained iPS cells can maintain pluripotency for a long time in in vitro culture and passage, solving the problem of traditional iPS cells. The reprogramming method has the defects of low reprogramming efficiency and easy differentiation during passage.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an induced pluripotent stem cell and its preparation method and application. Background technique [0002] Induced pluripotent stem cells (iPS cells) refer to stem cells with pluripotent differentiation potential obtained by introducing pluripotent genes into somatic cells or reprogramming genes under the action of other inducing factors. In August 2006, the Yamanaka research group of Kyoto University in Japan transferred the four genes Oct4, Sox2, Klf4 and c-Myc (OSKM) into mouse fibroblasts, and for the first time directly reprogrammed somatic cells into embryonic stem cell-like pluripotent cells. Stem cells, marking the birth of somatic cell reprogramming into iPS cell technology. At the end of 2007, Yamanaka's group and Thomson's group successfully reprogrammed human fibroblasts into iPS cells. This study has greatly promoted the research and application of human stem cells. It...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10
CPCC12N5/0653C12N5/0655C12N5/0657C12N5/0696C12N15/86C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2501/998C12N2506/1307C12N2506/45C12N2510/00C12N2740/15043C12N2800/107
Inventor 李玮
Owner HAINAN YILING MEDICAL INDUSTRY & DEVELOPMENT CO LTD
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