Human source chromosome targeting vector of human beta-globin gene cluster and application therefor

A technology of targeting vectors and globin, applied in gene therapy, introduction of foreign genetic material using vectors, blood diseases, etc., can solve the problems of unstable gene integration, low expression level, random integration sites of viral vectors, etc., to avoid safety Sexual problems, the effect of avoiding immune responses

Inactive Publication Date: 2010-04-28
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Early viral vector-mediated globin gene transfer studies found that the expression level of the globin gene carrying only proximal regulatory elements was extremely low in mice, and the viral vector itself still had problems such as random integration sites and unstable gene integration. ; More importantly, all current drugs for β-thalassemia gene therapy only transfer a single functional gene in the globin gene cluster and some of the necessary regulatory elements upstream, resulting in a low expression level of the transferred gene

Method used

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  • Human source chromosome targeting vector of human beta-globin gene cluster and application therefor
  • Human source chromosome targeting vector of human beta-globin gene cluster and application therefor
  • Human source chromosome targeting vector of human beta-globin gene cluster and application therefor

Examples

Experimental program
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Embodiment 1

[0044] Example 1. Construction and Identification of Human Chromosomal Targeting Vector of Human β-globin Gene Cluster

[0045] The homology analysis and chromosome location of the two homology arms TGLS1 and TGLS2 in the human chromosome BAC targeting vector BACMS are carried out. The chromosome location map is as follows: figure 2 As shown (A in the figure indicates that it is located at 21p11, and B indicates that it is located at 7q36.2), indicating that they are not only highly homologous to the human melanoma antigen gene (B melanomaantigen, BAGE) on the short arm of the D and G chromosomes, but also related to human The Homo sapiens myeloid / lymphoid or mixed-lineage leukemia 3 (MLL3) gene at 7q36.2 at the end of the long arm of chromosome 7 is highly homologous, and the sequences of TGLS1 and TGLS2 are the last inclusion of the MLL3 gene The homology of the gene is 97%. Therefore, it is believed that using this vector to construct a new human chromosome-targeting vecto...

Embodiment 2

[0059] Embodiment 2, the acquisition of monoclonal cell line and the structural analysis of genomic DNA thereof

[0060] 1. Obtaining monoclonal cell lines

[0061] The human-derived chromosomal BAC targeting vector (BACMS / BGGC) constructed in Example 1 containing the complete β-globin gene cluster was transfected into K562 cells by the liposome method (or the BACMS / BGGC was treated with the restriction enzyme SgrAI K562 cells were electroporated after linearization). In order to prevent the possible loss of positive cell clones under the simultaneous action of two screening drugs, G418 (purchased from Sigma) and GCV (purchased from Sigma), a step-by-step screening method was adopted. After screening with 400 μg / mL G418 for 6 weeks, 12 monoclonal cell lines resistant to G418 were obtained, the genomes were extracted and each cell line was frozen; at the same time, 10 μg / mL GCV was used for negative selection of 12 cell lines. As a result, two cell lines were obtained, named ...

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Abstract

The invention discloses a human-source chromosome targeting carrier and preparing method and appliance of beta-globin gene cluster, which is characterized by the following: the carrier contains recombinant human-source chromosome targeting BAC carrier of Beta-globin gene cluster,BGGC, whose GenBank number is NG_000007 to construct carrier as human-source chromosome targeting BAC carrier BACMS.

Description

technical field [0001] The invention relates to a gene targeting carrier and its application, in particular to a human chromosome targeting carrier of human β-globin gene cluster, its preparation method and its application in the preparation of β-thalassemia therapeutic drug. Background technique [0002] The human globin gene family is divided into two gene clusters, α and β. The human α-globin gene cluster located in the proximal telomeric region of chromosome 16 (16p13.3) is arranged in the order of 5′-ζ2-φζ1-φα2-φα1-α2-α1-θ1-3′, with a total length of about 40kb. Among them, ζ is an embryonic functional gene, and α is a fetal and adult functional gene. The human β-globin gene cluster located in the p15.5 region of chromosome 11 is arranged in the order of 5′-ε-Gγ-Aγ-ψβ-δ-β-3′, with a total length of about 75kb, of which ε is an embryonic functional gene , Gγ and Aγ are fetal-type functional genes, and δ and β are adult-type functional genes. At different stages of dev...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/85A61K48/00A61P7/00
Inventor 周海胜赵娜刘德培夏昆夏家辉
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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