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Efficient genetic-modification-free iPSC induced and industrialized monoclonal picking platform and application

A technology of SOX2 and OCT4, applied in the field of biomedicine, can solve the problems of poor safety, complex operation and low efficiency of iPSC, and achieve the effect of reducing carcinogenicity, high induction efficiency and improving operability

Active Publication Date: 2021-10-01
CHENGNUO REGENERATIVE MEDICINE TECH (ZHUHAI HENGQIN NEW AREA) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are various induction methods for iPSCs, but there are the following technical defects: (1) iPSC-based autologous / allogeneic cell therapy requires a stable, reproducible, gene-editing-free induction method, and animal-derived The current commonly used iPSC induction methods are mainly based on gene integration and animal-derived medium; (2) The application of cell banks, disease modeling and cell therapy has put forward higher and higher requirements for hiPSC technology, among which Including the requirements for safety, while the current commonly used iPSC induction methods usually use a large number of reprogramming factors, including OCT4 (POU5F1), SOX2, L-MYC / l-MYC, KLF4, LIN28, NANOG, SV40LT, and more The use of quantitative reprogramming factors leads to poor safety of reprogrammed iPSCs; (3) The commonly used iPSC induction method usually uses physical means to obtain single clones in the stage of isolating single clones, which cannot guarantee that the obtained clones are single clones. Cell cloning may also introduce undifferentiated cells, which is complicated and inefficient

Method used

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  • Efficient genetic-modification-free iPSC induced and industrialized monoclonal picking platform and application
  • Efficient genetic-modification-free iPSC induced and industrialized monoclonal picking platform and application
  • Efficient genetic-modification-free iPSC induced and industrialized monoclonal picking platform and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0137] The construction of embodiment 1 reprogramming plasmid

[0138] 1. Experimental materials

[0139]The pEGFP N1 plasmid described in this example was purchased from Addgene.

[0140] 2. Experimental method

[0141] (1) Use the pEGFP N1 plasmid as the backbone vector, and use EcoR1 and Not1 as restriction sites;

[0142] (2) Gene synthesis of OCT4-IRES-SOX2 and E6-E7, the synthesis company is Anhui General Biotechnology Co., Ltd., insert OCT4-IRES-SOX2 and E6-E7 between EcoR1 and Not1 of the pEGFP N1 plasmid, and the vector is named OCT4 -IRES-SOX2 and E6-E7;

[0143] (3) The sequence of OCT4-IRES-SOX2 is shown in SEQ ID NO: 1. OCT4 and SOX2 are connected sequentially through a spacer sequence, and the spacer sequence is IRES. The IRES sequence can use self-cleaving polypeptide 2A (Self-cleaving 2A peptide, 2A) Replacement, including E2A, P2A, T2A, F2A, etc.

[0144] (4) The sequence of E6-E7 is shown in SEQ ID NO: 2, E6 and E7 are sequentially connected by a spacer ...

Embodiment 2

[0145] Example 2 PBMC expansion preparation operation

[0146] 1. Experimental materials

[0147] Peripheral blood mononuclear cells (PBMC) were purchased from STEMCELL Technologies, the catalog number is #70072.2; StemSpan TM SFEM II was purchased from STEMCELL Technologies, part number #09655; StemSpan TM CD34+Expansion Supplement was purchased from STEMCELL Technologies, the product number is #02691.

[0148] 2. Experimental method

[0149] (1) Take out a frozen PBMC of 12-15 million from the liquid nitrogen tank, place it in a hot water bath with a water temperature of 37-40°C, shake it gently, and when only tiny crystals remain in the frozen tube, use Thoroughly disinfect the surface of the cryotube with 75% alcohol, wipe off the 75% alcohol with sterile gauze, and put it into the ultra-clean workbench;

[0150] (2) Use a pipette gun to add the cell suspension into 10 mL of hematopoietic stem cell basal medium preheated in advance, mix gently, take samples and count, ...

Embodiment 3

[0154] Example 3 PBMC electroporation operation

[0155] 1. Experimental materials

[0156] The reprogramming plasmids OCT4-IRES-SOX2 and E6-E7 constructed in Example 1 of the present invention, and the PBMCs cultured and expanded in Example 2 of the present invention.

[0157] 2. Experimental method

[0158] (1) After gently pipetting the cells to be electroporated (PBMC), collect the cell suspension into a new 50mL centrifuge tube;

[0159] (2) Wash the cell wells once with 2mL DPBS, transfer the cleaning solution to the above-mentioned 50mL centrifuge tube, mix gently and then take samples and count;

[0160] (3) Centrifuge after trimming, 200g, 10min, centrifuge and count;

[0161] (4) According to the counting results, the amount of seeded cells on the 6-well plate is 800,000-2,000,000 cells per well;

[0162] (5) After centrifugation, according to the cell volume, resuspend the cells to make the cell concentration 0.8×10 6 -2.0×10 6 cells / 100μL, after resuspension,...

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Abstract

The invention discloses an efficient genetic-modification-free iPSC induced and industrialized monoclonal picking platform and application. The platform can efficiently perform reprogramming and only needs to use a minimum number of reprogramming factors (OCT4, SOX2, E6 and E7). In a monoclonal separation stage, SSEA4 / TRA-1-60 is taken as a selection marker, a large number of single cell clones are obtained through a flow cytometry sorting technology. The platform has the advantages of being high in reprogramming efficiency, high in safety, easy to operate, capable of achieving large-scale production and the like.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a high-efficiency and non-genetic modification iPSC induction and industrial monoclonal picking platform and its application. Background technique [0002] In 2006, Takahashi and Yamanaka introduced several transcription factors into differentiated mouse skin fibroblasts, and obtained pluripotent stem cells similar to embryonic stem cells (Embryonic stem cells, ESCs), called "induced pluripotency Stem cells" (Induced pluripotent stem cells, iPSCs) (Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors [J]. Cell, 2006, 126(4): 663-676.), In 2007, Takahashi successfully obtained human iPSCs using human fibroblasts (Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J]. Cell, 2007, 131( 5):861-872.), in the same year, Yu and Thomson...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/10A61K35/545A61P35/00A61P37/06A61P43/00
CPCC12N5/0696A61K35/545A61P35/00A61P37/06A61P43/00C12N2510/00C12N2501/998C12N15/85C12N5/0018C12N2501/602C12N2501/603C12N2502/1171
Inventor 吴理达顾雨春
Owner CHENGNUO REGENERATIVE MEDICINE TECH (ZHUHAI HENGQIN NEW AREA) CO LTD
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