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Treatment method for preparing sperm of transgenic mouse

A treatment method and sperm technology, applied in the field of transgenic, can solve the problems of large fluctuations, contradictions, and great differences in transgenic efficiency, and achieve the effects of reliable and controllable transgenic results, improved permeability, and improved transgenic efficiency.

Inactive Publication Date: 2013-08-14
朱孝荣 +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0009] To sum up, the above-mentioned technology of using sperm as carrier to prepare transgenic mice is still very immature. It is extremely large, even contradictory, and the results are very random and uncertain, and many of the controllable links and transfection mechanisms are still worthy of experimental research

Method used

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  • Treatment method for preparing sperm of transgenic mouse
  • Treatment method for preparing sperm of transgenic mouse
  • Treatment method for preparing sperm of transgenic mouse

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, the linear DNA fragment containing target gene

[0025] Using adult C57BL / 6J mouse tail as experimental material, its genomic DNA was extracted and used as a template to design a specific gene according to the mouse tyrosine hydroxylase (TH) promoter DNA sequence (gene sequence number ×53503) in GenBank. PCR primers, since the sequence is 3650bp, using overlap extension PCR method, design 2 pairs of primers, respectively amplify the upstream 1870bp upstream sequence and the downstream 1800bp downstream sequence, and then overlap extension to amplify the whole fragment, its sequence is SEQ ID NO. 7.

[0026] THF1:5-CATGG ATCC AATGATTGGTATGGCTGGGGTCC-3 (SEQ ID No.1, introduced BamH I restriction site)

[0027] TH R 1:5-AGC AAGCTT AGCTGGTGGTCCCGAGTTCTGTC-3 (SEQ ID No.2, introduced HindIII restriction site)

[0028] THF2: 5-GTTGCTGACCCCAGGAAGAGTTC-3 (SEQ ID No. 3)

[0029] THR2: 5-GAACTCTTCCTGGGTCAGCAAC-3 (SEQ ID No. 4)

[0030] The PCR amplification of ...

Embodiment 2

[0036] Embodiment 2, sperm treatment and DNA co-incubation

[0037] Take the epididymis semen of the mice, and use a Pasteur pipette to draw the semen into the well-balanced HTF micro-droplet culture solution. Proceed as follows:

[0038] 2.1 DMSO treatment method: DMSO and DNA fragments were added to the semen, and the sperm concentration was adjusted to 10 7 At the same time, the concentration of DMSO in the semen was 2%, and the amount of pTH-CB linear fragment was 20 μg / ml. After adjustment, the semen was placed at 4°C for 10 minutes, and then diluted 10 times with HTF after equilibrium, at 37°C, 5% CO 2 Capabilize in the incubator for 1h.

[0039] 2.2 Direct incubation method: pTH-CB fragments were added to the semen at a concentration of 0.01-1ug / 10 6 Sperm, 37°C, 5% CO 2 Capabilize in the incubator for 1h.

[0040] 2.3 Liposome treatment method: 200uL semen (sperm concentration 7*10 5 pcs / ml) Lipofectamin TM2000Reagent0.5uL, 0.5ug pTH-CB fragment, 37℃, 5%CO 2 Cap...

Embodiment 3

[0042] Embodiment 3, in vitro fertilization

[0043] C 57 The cumulus of BL / 6J mice was transferred to another HTF medium, and the semen treated and capacitated by different methods in Example 2 were added respectively. Before adding the semen, the sperm viability after various treatment methods were detected. And adjust the concentration of sperm during fertilization to 7*10 5 pieces / ml. Fertilize for 5 hours in a 37°C, 5% CO2 incubator. After fertilization, the fertilized eggs were sucked out under the microscope, washed with HTF three times, and the fertilization situation was checked. Those with the second polar body were judged as fertilized eggs. Calculate the fertilization rate, see Table 1:

[0044] Table 1 Comparison of sperm motility and in vitro fertilization rate after four methods of treatment

[0045] Sperm processing method

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Abstract

The invention belongs to a transgenic field, and relates to a treatment method for preparing sperm of a transgenic mouse. The treatment method for preparing sperm of the transgenic mouse, comprises: incubating mouse epididymal sperm at 37 DEG C in a 5% CO2 incubator for 8-10min, mixing a linear DNA fragment containing a target gene into the sperm, ice-bathing the sperm at 2-5 DEG C for 25-30min, heat-shocking the sperm at 40-45 DEG C for 1-3min, ice-bathing the sperm at 2-5 DEG C for 3-5min, and then incubating the sperm at 37 DEG C in a 5% CO2 incubator for 50-70min for capacitation The method can raise permeability of a sperm membrane, is convenient for introducing a gene fragment into the sperm, and raises transgenic efficiency. The invention solves the problems of low efficiency, bad stability and difficult passage when sperm is used to prepare the transgenic mouse. An effective method for preparing the transgenic mouse is disclosed.

Description

technical field [0001] The invention belongs to the field of transgenics, and relates to a sperm treatment method for preparing transgenic mice. Background technique [0002] Transgenic methods have been widely used in various fields of modern life science research and gene function research. At present, gene transfer techniques mainly include DNA microinjection, ES cell-mediated method, retroviral vector method, sperm carrier method and so on. Although these methods can be used to introduce foreign genes into target cells and obtain corresponding transgenic animals, there are some limitations. Among them, the most commonly used DNA microinjection method requires expensive experimental equipment, cumbersome work, high technical requirements, low rate of exogenous gene introduction, and high production costs; although the gene transfer efficiency of the retroviral vector method is high, the gene insertion It is random, and the size of the DNA fragments carried is limited, a...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N5/10
Inventor 朱孝荣袁红华彭长凌
Owner 朱孝荣
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