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
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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.
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com