Transgenic vector system capable of promoting cell transplantation and gene expression and applications of transgenic vector system

A transgenic carrier and gene expression technology, applied in the direction of vectors, nucleic acid carriers, and the use of vectors to introduce foreign genetic material, etc., can solve problems such as hindering applications, uncommon characteristics of target genes, and restricting use, so as to achieve great application value and avoid drug toxicity the effect of the influence

Active Publication Date: 2017-03-08
上海百英生物科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently three types of in vivo screening methods: 1) The target gene has the ability to make the host cell continue to divide and promote survival, so that in vivo screening occurs naturally without any screening pressure, but the characteristics of this target gene are not common, That is, not all target genes have the ability to make the host cell continue to divide and promote survival; 2) In addition to the target gene, a screening gene is introduced at the same time, which enables the host cell to have drug-dependent ability to promote survival (so-called The cell growth switch cell growth switches), but this method shows that the screening only oc

Method used

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  • Transgenic vector system capable of promoting cell transplantation and gene expression and applications of transgenic vector system
  • Transgenic vector system capable of promoting cell transplantation and gene expression and applications of transgenic vector system
  • Transgenic vector system capable of promoting cell transplantation and gene expression and applications of transgenic vector system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. In vivo screening system using BCL2 mutant S70A as screening gene

[0039] In Example 1, the BCL2 mutant S70A was used as the screening gene, and the retroviral vector MIGR1 was used as the vector. As mentioned above, S70A retains part of the anti-apoptotic ability of BCL2, but removes its tumor-promoting ability, so it is particularly suitable as a natural screening gene in vivo.

[0040] (1) Insert S70A before the IRES of MIGR1, which is the MIGR1-S70A plasmid, such as figure 1 shown.

[0041] (2) The retroviral supernatant was prepared with MIGR1-S70A plasmid and packaging cell BOSC23. Specifically, BOSC23 cells were cultured in 6-well cell culture plates in complete medium (Dulbecco's Modified Eagle's Medium, DMEM) complete medium (adding 10% fetal bovine serum, 100 units / ml penicillin, 100 μg / ml streptomycin, 2mM L-glutamine, etc.) (37°C, 5% CO 2 ), when the cells are 90% confluent, replace the old medium with fresh DMEM medium containing 5% FBS and ...

Embodiment 2

[0048] Example 2. In vivo screening system based on induction system and lentiviral vector using Bcl-2 mutant S70A as screening gene

[0049] Lentiviruses have safer properties than retroviruses. Therefore repeat the research of embodiment 1 with self-inactivating feline immunodeficiency virus ( image 3 , the empty viral vector was from SBI Company). The packaging cells used for virus production are 293T cells. In addition to the lentiviral vector, two other packaging plasmids (pFIV-34N, pVSV-G) should be transferred into the 293T cells. The lentivirus packaging method, bone marrow cell acquisition, virus infection and cell transplantation are the same as in Example 1.

[0050] Six months after transplantation, it was found that lentivirus-based S70A could increase the proportion of GFP-positive cells, but unlike retrovirus S70A, lentivirus S70A could only increase the proportion of GFP-positive cells to a low level, about 30%. By comparing the fluorescence intensity value...

Embodiment 3

[0051] Example 3. Non-myeloablative pre-transplant conditioning by 5-FU treatment

[0052] In clinical practice, transplant recipient patients often cannot use lethal doses of radiation to irradiate myeloablation, so it is very critical whether non-myeloablative pre-transplantation treatment with drugs is feasible. Before transplantation, the recipient mice were injected with 150 mg / kg body weight of pentafluorouracil (5-FU). Six months after transplantation, it was found that this pretreatment was sufficient to successfully transplant bone marrow cells infected by retrovirus MIGR1-S70A ( Figure 4 ), so this method is very suitable for clinical application.

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Abstract

The invention discloses a transgenic vector system capable of promoting cell transplantation and gene expression. The transgenic vector system comprises a screening gene system, a target gene expression system, and a vector used for bearing the two systems, wherein the screening gene system is an expression system of a gene capable of promoting cell division or cell survival or a mutant of the gene, or a silencing system of a gene capable of inhibiting division or promoting cell death. Compared with the prior art, the system has the advantages that firstly, the limitation that whether the target gene has the growth vigor or not is eliminated, secondly, the screening occurs on both the stem cell level and the dedifferentiating cell level, and therefore, the gene modification cells can be stably maintained all the life or the number of the gene modification cells can be increased; thirdly, medicine screening is not needed, and therefore, the influences of the toxicity of the medicines are avoided; moreover, the mutant only promoting the cell survival but not promoting the cell mutation is adopted, and therefore, the risk of causing cancer is avoided. Therefore, the system has great application values in gene and cell therapy and the manufacturing of animal bioreactors or animal models based on adult (stem) cells.

Description

technical field [0001] The invention relates to a transgene carrier system, especially a carrier system capable of promoting cell transplantation and gene expression. Background technique [0002] Gene therapy or the establishment of adult (stem) cell-based animal bioreactors or animal models require stable target gene expression levels and stable target gene expression positive cell numbers in recipient patients or animals. However, in reality, it generally occurs that the efficiency of expressing the target gene in a single cell decreases and the proportion of cells expressing the target gene decreases. An approach to address the above deficiencies is to perform in vivo screening. There are currently three types of in vivo screening methods: 1) The target gene has the ability to make the host cell continue to divide and promote survival, so that in vivo screening occurs naturally without any screening pressure, but the characteristics of this target gene are not common, ...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
CPCC12N15/85C12N15/86C12N2740/11043C12N2740/15043C12N2800/30C12N2830/36
Inventor 王彦刈马珊
Owner 上海百英生物科技股份有限公司
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