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A method for constructing bone marrow cell drp1 gene-specific knockdown mouse model

A bone marrow cell and mouse model technology, applied in other methods of inserting foreign genetic materials, genetic engineering, plant genetic improvement, etc., can solve the problems of low tissue specificity, high economic cost, long experimental period, etc., and achieve success. High rate, ensure interference efficiency, and reduce the effect of virus dosage

Active Publication Date: 2021-08-27
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

At present, bone marrow-specific gene model animals mainly rely on Cre-LoxP gene recombination technology, which is obtained by crossing bone marrow-specific Cre recombinase-expressing mice and Loxp transgenic mice. The main disadvantages are long experimental period and high economic cost. , heavy workload, and low organizational specificity

Method used

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  • A method for constructing bone marrow cell drp1 gene-specific knockdown mouse model
  • A method for constructing bone marrow cell drp1 gene-specific knockdown mouse model
  • A method for constructing bone marrow cell drp1 gene-specific knockdown mouse model

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Experimental program
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Embodiment 1

[0045] 1. Preparation of Drp1 gene knockdown lentiviral vector

[0046] Drp1 primer design is shown in Table 1:

[0047] Table 1 Drp1 primers

[0048]

[0049] By annealing, double-stranded shRNA is produced. Digest vector Lenti-H1-shRNA-puro vector (restriction site EcoRI, BamHI) to linearize it. Double-stranded shRNA ligated linearized vector. After the competent cells were transformed, the plate was spread, and the clones were picked and sequenced. The vectors with correct sequencing were amplified, the plasmids were extracted to package lentiviruses, and the Drp1-shRNA lentiviral vectors were obtained, and the empty vector (EmptyVector) was used as a control.

[0050] 2. Screening of the number of bone marrow transplanted cells and MOI of infected virus

[0051] 2.1 Quantitative screening of bone marrow transplantation cells

[0052] (1) Bone marrow cell isolation method: After the C57BL / 6-GFP mice were killed by neck dislocation, the tibia and femur of the limbs ...

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Abstract

The invention provides a method for constructing a bone marrow cell Drp1 gene-specific knockout mouse model, and relates to the field of biotechnology. The preparation method comprises the following steps: (1) construction of Drp1-shRNA lentiviral vector; (2) bone marrow Lentivirus infection of cells in vitro; (3) Implantation of infected cells into mice to establish a model. The present invention takes the lead in constructing a bone marrow cell Drp1 gene-specific knockout mouse model by using the inner canthus intravenous injection method, and optimizes the number of transplanted bone marrow cells and virus infection conditions, and has the advantages of rapid modeling, high success rate, good specificity and The advantage of low economic cost. It not only provides an animal model for studying the immunological function of Drp1 gene, but also provides a new basis for the preparation of other bone marrow-specific gene model animals.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a bone marrow cell Drp1 gene-specific knockout mouse model. Background technique [0002] In recent years, research on immunity and disease has become an international hotspot, especially in the fields of tumors, type 2 diabetes, and cardiovascular and cerebrovascular diseases. Research on immune cell functions has increased year by year. Bone marrow is the hematopoietic tissue in the body. It is mainly distributed in the bone cavity and cancellous bone of long bones. It can generate various stem cells and plays a vital role in maintaining hematopoietic and immune functions. Immune cells derived from bone marrow mainly include T cells, B cells, NK cells, monocytes, and macrophages. Among them, T lymphocytes are pluripotent stem cells derived from bone marrow, which can mature in the thymus, and then have the function of fighting infection and killing tumor ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/90C12N15/12A01K67/027
CPCA01K67/0271A01K2207/12A01K2217/075A01K2227/105A01K2267/0387C07K14/47C12N15/86C12N15/907C12N2740/15043C12N2800/107
Inventor 于卫华海春旭闫文俊王欣李文丽刘江正
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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