Method for transduction of bone marrow mesenchyma stem cell by human telomerase gene

A bone marrow mesenchymal and stem cell technology, applied in the direction of using vectors to introduce foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problem of low immortalization rate of SV40 transfected cells, and achieve the effect of improving the success rate

Inactive Publication Date: 2007-10-31
ZHEJIANG UNIV
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Problems solved by technology

However, the results of Shay et al. (1989) showed that in most cas

Method used

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  • Method for transduction of bone marrow mesenchyma stem cell by human telomerase gene
  • Method for transduction of bone marrow mesenchyma stem cell by human telomerase gene
  • Method for transduction of bone marrow mesenchyma stem cell by human telomerase gene

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Embodiment 1

[0025] The technology of the present invention adopts Hpa I and Not I restriction endonucleases to cut pGRN145 plasmid, separate and purify through agar gel electrophoresis to obtain hTERT cDNA fragment, and adopt the same restriction endonuclease digestion human retroviral vector pLXSN vector The pLXSN-hTERT recombinant expression vector for highly expressing hTERT was constructed, as shown in Figure 1.

[0026] Specific steps are as follows:

[0027] 1. Mass preparation of pGRN145 plasmid (alkaline lysis method):

[0028] 1) Resuspend 500 ml of washed bacteria (containing pGRN145 plasmid) culture pellet in 18 ml of solution I (containing 50 mmol / L glucose, 25 mmol / L Tris.Cl (pH 8.0), 10 mmol / L liter EDTA (pH 8.0)).

[0029] 2) Add 2 ml of freshly prepared lysozyme solution [10 mg / ml, dissolved in 10 mmol / L Tris·Cl (pH 8.0)] (When the pH of the solution is lower than 8.0, lysozyme cannot work effectively.)

[0030] 3) Add 40 ml of newly prepared solution II [0.2 mol / L NaOH...

Embodiment 2

[0101] The technology of the present invention adopts the LipofectAMINE liposome transduction kit, and carries out the preparation of the recombinant expression carrier suspension and the transduction of human bone marrow mesenchymal stem cells according to the usage method of the kit. According to the instructions of the kit, the amphagocytic packaging cell line PA317 was used to prepare the recombinant virus vector suspension, and the recombinant expression vector was screened with 200 μg / ml hygromycin for 4 days. With the aid of 8 μg / ml of 1,5-dimethyl-1,5-diazaundecamethylene polymethyl bromide (Polybrene; Sigma, St. Louis, MO), in a 10-cm Petri dish (Maxisorp, Nunc, Denmark) pair 2.0×10 5 Individual BMSCs were transduced with recombinant expression vectors for 8 hours. Cells were then washed with phosphate-buffered saline (PBS), and cells were first selected with 20 μg / ml hygromycin for 7 days. After 7 days, the cells were washed with normal culture medium, and the cell...

Embodiment 3

[0103] The technology of the present invention adopts RT-PCR technology and Northern blotting technology to detect the expression level of hTERT gene in transduced human bone marrow mesenchymal stem cells. The results showed that the bone marrow mesenchymal stem cells transduced with the hTERT gene effectively expressed the hTERT gene, see Figure 2A, 2B, the figure shows (A) RT-PCR was used to detect the expression of the hTERT gene, including the Marker (3kb) swimming lane, Lane 1: expression of hTERT gene and β-actin gene in hTERT-hMSCs at passage 95, lane 2: expression of hTERT gene and β-actin in hTERT-hMSCs at passage 275, lane 3: normal hMSCs at passage 13 Expression of hTERT gene and β-actin gene in cells, lane 4: RT-PCR with water as control. (B) Detection of hTERT gene expression by Northern blotting, including lane 1: expression of hTERT gene and β-actin gene in the 95th passage hTERT-hMSCs cells, and lane 2: hTERT gene and β-actin gene expression in the 275th passag...

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Abstract

The invention discloses a method to transduce full material stem cell among marrow with human end particle enzyme gene, which comprises the following steps: enzyme-cutting pGRN145 plasmid with Hpa I and Not I restriction enzymes; getting hTERT cDNA fragment; connecting to pLXSN carrier with the same restriction enzymes; constructing pLXSN-hTERT retooling expressing carrier; using LipofectAMINE liposome transduction agent box; preparing pLXSN-hTERT retooling expressing carrier floated liquid through compatible eating property incasing line PA317; proceeding transduction for full material stem cell among marrow; constructing the stem cell with high effective expressing exogenesis hTERT gene; double-sieving with low density homomycin; getting the stem cell. This invention can reach the goal to increase life cycle of full cytoplasma stem cell among marrow.

Description

technical field [0001] The invention belongs to biological technology and relates to a new technology for constructing and screening bone marrow mesenchymal stem cells exogenously expressing human telomerase. This technique enables bone marrow mesenchymal stem cells to highly express exogenous human telomerase, thereby improving the life cycle of bone marrow mesenchymal stem cells. Background technique [0002] At present, the commonly used technologies that can be used to prolong the life cycle of cells and make cells immortal can be roughly classified into four types: Epstein-Barr virus (EBV) infection technology, simian virus 40 (SV40) large T antigen technology, and human papilloma type 16 Virus (HPV16) E6 / E7 technology, telomerase gene technology. Co-cultivation of Epstein-Barr virus and cells to immortalize cells is the most widely used B lymphocytes. One of the most notable features of EB virus immortalized B lymphocytes is the increase in telomerase activity, which...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/52
Inventor 王金福黄国平
Owner ZHEJIANG UNIV
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