Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Establishment method for differentiating swine germline stem cells into sperms in vitro

A technology of reproductive stem cells and establishment methods, applied in the direction of reproductive cells, biochemical equipment and methods, animal cells, etc., can solve problems such as low success rate and poor operability, and achieve simple operation and good repeatability

Inactive Publication Date: 2016-06-01
SOUTH CHINA AGRI UNIV
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the research is carried out on humans and mice, but there are still some phenomena with low success rate and poor operability, and there are not many studies on sperm differentiation in large animals.

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
  • Establishment method for differentiating swine germline stem cells into sperms in vitro
  • Establishment method for differentiating swine germline stem cells into sperms in vitro
  • Establishment method for differentiating swine germline stem cells into sperms in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 A kind of establishment method of porcine mGSCs differentiated sperm in vitro

[0031] see figure 1 , is the roadmap for establishing in vitro sperm differentiation of porcine mGSCs in this embodiment, and the method for establishing sperm in vitro in vitro differentiation of porcine mGSCs in this embodiment includes the following specific steps:

[0032] 1) Construction of in vitro induced sperm differentiation "Niche" feeder layer

[0033] The establishment of a balanced and stable "Niche" feeder layer in vitro is the guarantee to promote the differentiation of porcine mGSCs into sperm in vitro. The testis tissue of Landrace piglets was isolated and cultured, and primary piglet testis tissue fibroblasts (TFCs) (removed germ cells) were obtained by enzymatic digestion and wall-attachment methods, using a specific medium (15% FBS, 55 μM β-mercaptoethanol, 1 %ITS, 1% L-glutamine, 1% nucleotides, 1ng / mLLH, 500ng / mlFSH and DMEM medium containing 1% Penicilin...

Embodiment 2

[0036] Example 2 Detection of the meiotic process of inducing mGSCs to differentiate into sperm in vitro

[0037] The meiosis protein SYCP3 plays an important role in the process of meiosis, and is a common detection protein for identifying the process of meiosis. By detecting the induced differentiation of porcine mGSCs, it was found that meiosis occurred, and SYCP3 was distributed on the chromosomes, prompting its Completed the meiotic process, while no SYCP3 was detected in the control group ( Figure 4 ),in Figure 4 A is SYCP3 protein staining; Figure 4 B is Hoechst33342 stained nuclei; Figure 4 C is the Merge result of cells.

Embodiment 3

[0038] Example 3 Plotype analysis of in vitro induced spermatozoa

[0039] mGSCs entered meiosis under the culture conditions of the in vitro induced differentiation "Niche" feeder layer, and analyzed by flow cytometry, it was proved that they produced haploid sperm, showing that the DNA content was halved (such as Figure 5 B); Consistent with the result of the control group of pig semen (as Figure 5 C); while mGSCs and "Niche" feeder cells exhibited diploid cells with twice the DNA content of induced sperm and porcine semen (e.g. Figure 5 A).

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 invention discloses an establishment method for differentiating swine germline stem cells into sperms in vitro. The establishment method includes the steps of subjecting piglet testis tissues to isolation culture to obtain primary piglet testis tissue fibroblasts and germline stem cells, culturing the testis tissue fibroblasts for 15 days by means of a Niche culture medium for inducing in-vitro differentiation of sperms to obtain a Niche feeder layer for inducing in-vitro differentiation of sperms, and inoculating the germline stem cells to the Niche feeder layer to culture for 27-30 days, thereby obtaining haploid sperm-like cells in vitro by cytobiology analysis. The establishment method is easy to operate and good in repeatability, is an important breakthrough in research on swine mGSCs(male germline stem cells) in-vitro differentiation, provides an important basic technology for researches on swine embryonic in-vitro operation and transgenic breeding, and meanwhile, has high reference value in research on in-vitro differentiation of sperms of other species and assisted reproduction of human beings.

Description

technical field [0001] The invention belongs to the technical field of adult stem cell research, and more specifically, the invention relates to a method for establishing spermatozoa differentiated from porcine reproductive stem cells in vitro. Background technique [0002] Male germline stem cells (mGSCs) are a special kind of adult stem cells. They exist in the testis tissue, maintain a constant number, and can continuously differentiate to produce sperm. It is one of the main contents of reproductive development research. [0003] The orderly differentiation of sperm is the key to maintaining the fertility of male animals. In vitro studies on inducing mGSCs and other types of stem cells to differentiate into sperm have been carried out for many years, but there are few reports on the successful establishment of in vitro induction of mGSCs to develop into functional sperm. At present, most of the researches are carried out on humans and mice, but the success rate is still ...

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 Applications(China)
IPC IPC(8): C12N5/076
CPCC12N5/061C12N2500/32C12N2500/40C12N2500/44C12N2500/84C12N2501/31C12N2506/04
Inventor 白银山张守全朱翠冯美莹卫恒习李莉
Owner SOUTH CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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