Method for transfecting spermatid with lentiviral vector

A technology of lentiviral vector and sperm cells, which is applied in the field of transfection of Ningxiang Huapig sperm cells with lentiviral vector containing human SLC3A2 gene

Inactive Publication Date: 2017-01-04
NANHUA UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is the efficiency of the exogenous gene SLC3A2 gene brought into sperm cells, the optimization screening of the best

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for transfecting spermatid with lentiviral vector
  • Method for transfecting spermatid with lentiviral vector
  • Method for transfecting spermatid with lentiviral vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0079] PCR method to amplify SLC3A2 gene: oligo design, the target gene SLC3A2 sequence fragment is designed according to the number NM_001012662.2, the upstream and downstream primers are respectively added with NotI and BamHII and protective bases, used for subcloning of the vector, the base sequence of the human SLC3A2 gene as follows:

[0080]

[0081]

[0082]Primers were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0083] Dissolve oligo to 50μM, take the same volume of oligo into a 1.5ml centrifuge tube, mix well, and make oligomix.

[0084] Use the prepared oligo mix for the first round of PCR reaction, the PCR system is as follows:

[0085]

[0086] Reaction conditions

[0087]

[0088] The second round of PCR reaction was carried out with Y164-1 and Y164-62, and the template was the product of the first round of PCR reaction. The PCR system is as follows:

[0089]

[0090] Reaction conditions

[0091]

[0092]

[0093] T...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Titeraaaaaaaaaa
Mean titeraaaaaaaaaa
Login to view more

Abstract

The invention relates to a method for efficiently transfecting the spermatid of a Ningxiang spotted pig with a lentiviral vector containing a human SLC3A2 gene. The method comprises the following steps: amplifying the human SLC3A2 gene by using a PCR method; connecting the sequence fragment of the target gene SLC3A2 to a LV5 vector by using genetic engineering technology so as to construct a gene recombinant vector; and subjecting the lentiviral vector containing the human SLC3A2 gene and the spermatid of the Ningxiang spotted pig to incubation. An immunofluorescence method and a semi-quantitative PCR method are employed for detecting exogenous gene transferring efficiency of incubation of the lentiviral vector and spermatid under different conditions; and a microscopic method is used for detecting the vitality of the spermatid of lentiviral vector-spermatid coincubation under different conditions.

Description

technical field [0001] The invention relates to the fields of DNA genetic engineering and miniature pig transgenic engineering, in particular to a method for transfecting Ningxiang Huapig sperm cells with a lentiviral vector containing human SLC3A2 gene. Background technique [0002] Since the sperm carrier method is used to prepare transgenic animals, it has the characteristics of simple operation and low cost; the natural characteristics of sperm spontaneously absorbing foreign DNA can keep the foreign gene in good integrity. Lentiviral vectors use viruses to integrate genes into the host cell genome and stabilize heredity. Studies indicate that in many species, genetic modification using SMGT is more cost-effective than microinjection. However, the overall reproducibility of this technique is not ideal, the uptake rate of exogenous DNA by sperm and the rate of transgene expression in offspring are not high. It is difficult to solve the technique of co-incubating sperm w...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/867C12N15/12C12N5/10
Inventor 王宗保肖国华郑兴佘美华郑曲通丁婧璇姚艳张昕
Owner NANHUA UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap