A method for improving the developmental efficiency of porcine somatic cell nuclear transfer embryos

A technology of cell nuclei and embryos, applied in botany equipment and methods, animal cells, vertebrate cells, etc., can solve the problem of low overall efficiency of pig somatic cell cloning, improve the development rate of blastocysts, increase the number of blastocyst cells, The effect of improving the overall efficiency

Active Publication Date: 2019-08-09
南宁壮博生物科技有限公司
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although after two decades of development, the cloning technology has been relatively mature, and there is a set of overall technologies including oocyte collection, culture, donor cell isolation, culture, egg activation, embryo in vitro culture and embryo transfer, but so far, The overall efficiency of pig somatic cell cloning is still very low, about 1%

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
  • A method for improving the developmental efficiency of porcine somatic cell nuclear transfer embryos
  • A method for improving the developmental efficiency of porcine somatic cell nuclear transfer embryos
  • A method for improving the developmental efficiency of porcine somatic cell nuclear transfer embryos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the in vitro maturation culture of porcine oocyte

[0052] Take the pig ovary from the slaughterhouse, wash it three times with normal saline containing 100U / mL penicillin and 100U / mL streptomycin, and then use a 10mL disposable syringe and a 18-gauge needle to extract follicles with a diameter of 3-6mm Cumulus-oocyte complexes (COCs) wrapped with 3-5 layers of cumulus cells were selected from the liquid. COCs were rinsed twice with normal saline containing 100U / mL penicillin and 100U / mL streptomycin, then transferred to IVM solution, at 39°C, saturated humidity, 5% CO 2 Cultivate in the incubator for 42h.

Embodiment 2

[0053] Embodiment 2, the separation and cultivation of pig fetal fibroblast

[0054] Pig fetal fibroblasts were prepared according to the following steps in turn:

[0055]1. Take out the fetus from the uterus of pregnant sows at gestation day 35, wash with DPBS buffer solution containing 100U / mL penicillin and 100U / mL streptomycin.

[0056] 2. Take the fetus obtained in step 1, use ophthalmic scissors to remove the head, limbs and viscera in the ultra-clean workbench, wash the remaining part with DPBS buffer, cut it into pieces with sterilized ophthalmic scissors, and transfer it to a 100mm petri dish. 37°C, saturated humidity, 5% CO 2 Incubate for 4-6 hours in an incubator.

[0057] 3. After completing step 2, add DMEM culture solution containing 15% (volume percentage) fetal bovine serum to the culture dish, and heat the culture medium at 37°C, saturated humidity, and 5% CO 2 Subcultured in an incubator until the cell confluency reached 90%. The culture medium used for s...

Embodiment 3

[0059] Embodiment 3, the effect of chaetocin on pig fetal fibroblasts

[0060] 1. The pig fetal fibroblasts in the logarithmic growth phase prepared in Example 2 were inoculated to a 96-well plate (5 × 10 per well). 4 cells) were cultured statically at 37°C in DMEM medium containing 15% (volume percent) fetal bovine serum until the cells adhered to the wall.

[0061] 2. After completing step 2, take the 96-well plate and perform the following grouping process:

[0062] Drug group 1: discard the culture supernatant, add DMEM culture solution containing 20nM chaetin (15% fetal bovine serum + 100U / mL ampicillin + 100U / mL streptomycin sulfate), and let stand at 37°C for 24 hours;

[0063] Drug group 2: discard the culture supernatant, add DMEM culture solution containing 40nM chaetin (15% fetal bovine serum + 100U / mL ampicillin + 100U / mL streptomycin sulfate), and let stand at 37°C for 24 hours;

[0064] Drug group 3: discard the culture supernatant, add DMEM culture solution co...

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

No PUM Login to view more

Abstract

The present invention discloses a method for improving porcine somatic cell nucleus transplantation embryo development efficiency, and provides applications of chaetocin, wherein the applications comprise at least one selected from (a1) preparation of a nucleus donor cell culture liquid for animal somatic cell nucleus transplantation, (a2) preparation of a reconstruction embryo culture liquid for animal somatic cell nucleus transplantation, (a3) culture of a nucleus donor cell for animal somatic cell nucleus transplantation, (a4) culture of a reconstruction embryo for animal somatic cell nucleus transplantation, and (a5) promotion of the formation of blastula from the reconstruction embryo in animal somatic cell nucleus transplantation. According to the present invention, the nucleus donor cell or reconstruction embryo in animal somatic cell nucleus transplantation is treated with the small molecule drug chaetocin, such that the occurrence of the somatic cell nucleus transplantation re-programming is promoted, the blastula development and the blastula cell number of the cloned embryo are significantly improved, and the whole efficiency of the porcine somatic cell nucleus transplantation technology is improved; and the method provides important significance for the improvement of the whole efficiency of the porcine somatic cell nucleus transplantation technology.

Description

technical field [0001] The invention relates to the fields of embryo development and animal biotechnology, in particular to a method for improving the development efficiency of pig somatic cell nuclear transfer embryos. Background technique [0002] The technology of somatic cell nuclear transfer was born in 1997, and it was first successfully used in sheep. The main method is to transfer fully differentiated nucleated donor cells into enucleated oocytes to form reconstructed embryos through micromanipulation and cell fusion technology, and then activate the reconstructed embryos for culture and transplantation, and finally obtain a complete individual. Because the obtained individual has the same genotype as the donor cell, this technique is also called somatic cell cloning technique. Due to its unique technical advantages, somatic cell cloning technology has great application value in animal breeding, disease model construction, endangered animal protection, therapeutic c...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/877C12N5/071
CPCC12N5/0602C12N15/8778C12N2500/74
Inventor 李奎刘志国牟玉莲郑新民毕延震
Owner 南宁壮博生物科技有限公司
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