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Selecting method of gene participating in transfection of exogenous DNA from sperm

A screening method and gene technology, applied in the field of gene screening involved in sperm transfection of foreign DNA, can solve problems such as disease, normal immunological dysfunction, and destruction

Inactive Publication Date: 2015-04-29
赵永聚
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when the normal immunological function of the CD4 molecule is dysfunctional or destroyed, it can lead to disease

Method used

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  • Selecting method of gene participating in transfection of exogenous DNA from sperm
  • Selecting method of gene participating in transfection of exogenous DNA from sperm
  • Selecting method of gene participating in transfection of exogenous DNA from sperm

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specific Embodiment approach 1

[0015] Specific embodiment one: this embodiment participates in the gene screening method of sperm transfection exogenous DNA, carries out gene screening according to the following steps:

[0016] 1. Construction of recombinant plasmid pGB-mCD4;

[0017] 2. cDNA library screening to obtain the gene to be tested;

[0018] 3. Extract the yeast plasmid and amplify the positive clone of the gene to be tested;

[0019] 4. Plasmid pGB-mCD4 and the positive cloned yeast plasmid of the gene to be tested were co-transformed into yeast two-hybrid strain Y190;

[0020] 5. Carry out β-galactosidase color reaction, and the result of color development that turns blue is positive, and analyzed to obtain the genes involved in sperm transfection with exogenous DNA.

[0021] The β-galactosidase color test of the recombinant plasmid pGB-mCD4 did not turn blue and was negative, such as figure 2 shown. Use the bait vector pGB-mCD4 and the gene plasmid to be tested to co-transform the yeast two...

specific Embodiment approach 2

[0023] Specific embodiment two: the difference between this embodiment and specific embodiment one is: the recombinant plasmid pGB-mCD4 in step one can be constructed according to the following steps:

[0024] ① Obtaining the target fragment:

[0025] Total RNA was extracted from SPF grade Kunming mouse testis tissue, the first strand of cDNA was reverse transcribed, and PCR reaction was performed. The PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2; the PCR primers are forward primer and reverse primer,

[0026] The forward primer sequence is 5'-GGCCAATCCGGCC-3',

[0027] The reverse primer sequence is 5'-GGCCTCTAAGGCC-3';

[0028] Table 1

[0029] CD4 template (100ng / μL plasmid)

2.00 μL

4dNTP (10mmol)

1.00μL□

10×PCR Buffer

5.00μL□

MgCl 2 (25mmol)

5.00μL□

Forward primer (10~12μmol)

4.00μL□

reverse primer (10~12μmol)

4.00μL□

5M betaine

10.00μL□

...

specific Embodiment approach 3

[0035] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that in Step 2, MouseTestis MATCHMAKER cDNA library is used for screening. Other compositions and connections are the same as those in Embodiment 1 or 2.

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Abstract

The invention discloses a selecting method of gene participating in transfection of exogenous DNA from sperm, relates to a method for screening a gene participating in transfection of exogenous DNA from sperm, and aims at providing the method for screening the gene participating in transfection of the exogenous DNA from the sperm. The method comprises the following steps: 1 building a recombinant plasmid pGB-mCD4; 2 screening from a cDNA library to obtain a to-be-tested gene; 3 positively cloning the to-be-tested gene, extracting a yeast plasmid and amplifying; 4 transferring the plasmid pGB-mCD4 and the positively cloned yeast plasmid of the to-be-tested gene into a double-hybrid strain Y190 of the yeast; and 5 carrying out beta-galactosidase chromogenic reaction, if the chromogenic result becomes blue, judging to be positive, and analyzing, thereby obtaining the gene participating in the transfection of the exogenous DNA from the sperm. Through the method disclosed by the invention, the gene for transferring the exogenous DNA together with CD4 can be greatly and rapidly obtained; a new idea is developed for research on the sperm-mediated gene transfer mechanism; and a foundation is laid for building of simple, stable and efficient sperm-mediated genetically modified methodology.

Description

technical field [0001] The invention relates to a gene screening method involved in sperm transfection with exogenous DNA. Background technique [0002] CD4 molecule is a kind of single-chain transmembrane glycoprotein with a relative molecular mass of about 55 000 (units), which belongs to the immunoglobulin superfamily and is mainly expressed in some T cells, thymocytes, some B cells and B cells transformed by Epstein-Barr virus. Cells, monocytes-macrophages, and brain cell surfaces in specific regions. It plays an important role in the process of T cell development, signal transduction, intercellular communication, cellular immune response and humoral immune response. However, when the normal immunology of the CD4 molecule is dysfunctional or destroyed, disease may result. Studies in recent years have shown that gp120, the envelope glycoprotein of HIV-1, binds to the CD4 receptor on the surface of T cells, mediating the entry of the virus into human cells and causing cy...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6809
Inventor 赵永聚
Owner 赵永聚
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