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Method for rejecting B2M (beta 2-microglobulin) gene segment

A technology of gene fragments and microglobulins, applied in the field of transgenics, can solve the problems of time-consuming and laborious, and achieve the effect of low immunogenicity

Inactive Publication Date: 2015-05-13
SHENZHEN KEHUIRUI BIOLOGICAL MEDICINE CO LTD
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Gene targeting using homologous recombination is still a widely used technique to allow selected DNA sequences to be modified in a predetermined manner, but has the disadvantage of being time-consuming and labor-intensive

Method used

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  • Method for rejecting B2M (beta 2-microglobulin) gene segment
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  • Method for rejecting B2M (beta 2-microglobulin) gene segment

Examples

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Embodiment 1

[0039] Embodiment 1: the deletion of B2M in the human HEK293 cell

[0040] The present invention designs a 20-nucleotide mediator sequence, replaces a preset position of exon 2 of the human B2M gene with bases, and manufactures the required B2M targeting CRISPR / Cas 9 vector ( figure 1 A). In the presence of this mediator sequence, the cleavage efficiency of the CRISPR / Cas 9 system expressed as a plasmid vector or a baculovirus vector was evaluated in a T7E1 measurement assay in human fetal kidney (HEK) 293 cells. Cells were co-transfected with two plasmid vectors expressing Cas9 / tracrRNA and B2M crRNA or co-transduced with BV-Cas9 / tracrRNA and BV-B2M-crRNA (BV-B2M-CRISPR). as figure 1 Shown in B, compared with a single PCR band of approximately 0.46 kb in the control group, the amplification rate after amplification of genomic DNA isolated from HEK293 cells transfected with the plasmid B2M-targeting CRISPR / Cas9 system or BV-B2M-CRISPR Small fragments of 0.18kb and 0.28kb ca...

Embodiment 2

[0041] Example 2: Deletion of B2M in human U87 cells

[0042] In order to select stable human cell clones whose B2M gene is disturbed by BV-B2M-CRISPR, the present invention has produced a BV-based donor vector, BV-B2M-eGFP, for the integration of selection cassettes at specific sites through homologous recombination into the B2M locus ( figure 2 A). U87 glioma cells, a human cell line that can be easily transduced by BV, were transduced with BV-B2M-CRISPR and selected with G418 for 3 weeks. Surviving cells became eGFP positive with stable eGFP expression lasting at least 3 months ( figure 2 B). PCR genotyping ( figure 2 C and D) were used to examine integration of the eGFP donor cassette at specific sites of the B2M locus. Another pair of gametes complementary to the native B2M allele and amplifying a 0.9 kb DNA fragment were used to identify B2M homozygous knockout U87 cell clones. PCR extension was performed for 40 seconds to allow only short fragments of the nativ...

Embodiment 3

[0043] Example 3: B2M knockout in human pluripotent stem cells

[0044] After demonstrating the effectiveness of BV-B2M-CRISPR in human U87 cells, the present inventors tested this system on human pluripotent stem cells H1hESC using its high transduction efficiency for baculovirus vectors. 72 hours after the completion of BV transduction, G418 selection was started to enrich for eGFP positive cells. eGFP-positive iPSC colonies subsequently continued to expand by mechanical selection during normal reculture. Pure eGFP-positive hESC colonies were obtained approximately 3 months after expansion ( Figure 4 A). The percentage of eGFP positive cells in these clones was 18.5% at day 20 and rose to 98.6% by day 100. Using the PCR genotyping method described above, the present invention obtained two hESC clones with homozygous deletion of B2M. Such as Figure 4 In C, a 1.4 kb amplification in clone 2.3 demonstrates integration of the donor cassette at the B2M locus, and a deletio...

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Abstract

The invention discloses a method for rejecting a B2M (beta 2-microglobulin) gene segment. The method comprises steps as follows: (1) constructing a CRISPR / Cas9 vector system; (2) introducing a target gene into a recipient cell by the aid of the constructed CRISPR / Cas9 vector system, and rejecting the B2M gene segment; (3) detecting and identifying the transfected recipient cell. The method is a new genetic engineering method for rejecting a human cell gene in a targeted manner. According to the method, a baculovirus vector is used as a transfer system, a specific CRISPR / Cas9 component with a B2M gene rejected is efficiently expressed in a human cell, B2M gene expression is effectively blocked, and expression of a cell surface type-I HLA (human leukocyte antigen) molecule is reduced, so that a low-immunogenicity human cell is produced.

Description

technical field [0001] The invention relates to the field of transgenic technology, in particular to a genetic engineering method for deleting β2-microglobulin gene fragments. Background technique [0002] β2-microglobulin (B2M) is a component of the human leukocyte antigen class Ⅰ molecule (HLA-I) complex, which is closely related to the expression of HLA-I molecules on the cell surface and the immunogenicity of cells. The human leukocyte antigen system (HLA) located on the short arm of chromosome 6 is the most polymorphic genetic system known so far. They encode type 1 and type 2 cell surface molecules. HLA class I molecules are expressed on the surface of all nucleated cells and serve to present peptides of intracellular origin and regulate innate and acquired immunity. The expression of HLA class I molecules on the cell surface depends on whether they can properly combine with β2-microglobulin (B2M) to form a complex. The molecular mass of human B2M is 11.6kDa, contai...

Claims

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Application Information

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IPC IPC(8): C12N15/866
Inventor 王朔朱海宝
Owner SHENZHEN KEHUIRUI BIOLOGICAL MEDICINE CO LTD
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