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

Method for constructing porcine naive embryonic stem cell line

An embryonic stem cell and construction method technology, applied in embryonic cells, animal cells, vertebrate cells, etc., can solve the problems of not conducting chimera experiments, affecting the efficiency of embryonic stem cell culture and line establishment, and non-uniformity.

Inactive Publication Date: 2018-07-24
NANJING MEDICAL UNIV
View PDF3 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Studies have shown that by transfecting reprogramming factors into the inner cell mass of porcine in vitro fertilization blastocysts through lentiviral vectors, and then inoculating them in the LIF culture system of 2i (CHIR99021 and KP), porcine naive embryonic stem cells can be obtained (Telugu BP, 2011, The Journal of biological chemistry286(33):28948-28953), but because of no chimera experiments, it cannot be identified as true naive embryonic stem cells
At the same time, due to the method of transfection of endoderm, different blastomeres are exposed to exogenous viruses and gene heterogeneity, which leads to the chimerism of gene integration between different cells of endoderm, which ultimately affects Embryonic Stem Cell Culture and Line Establishment Efficiency
[0004] At present, in terms of establishing naive embryonic stem cell lines using traditional methods, mice and rats have been successfully established, but porcine naive embryonic stem cells have not yet been able to establish stable cell lines

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 constructing porcine naive embryonic stem cell line
  • Method for constructing porcine naive embryonic stem cell line
  • Method for constructing porcine naive embryonic stem cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Obtaining of Transgenic Positive Pig Fetal Fibroblasts

[0053] The cryopreserved primary pig fetal fibroblasts were resuscitated in a 90 mm cell culture dish, cultured with cell culture medium (DMEM+20% FBS) until the cells grew to a confluence of about 90%, and the cells were collected. According to the nucleofection instructions, the primary porcine fetal fibroblasts were transfected with the U023 program using a Lonza nucleofection instrument. 48 h after transfection, the cells were digested with trypsin and 3 × 10 5 Cells were seeded in a 100 mm culture dish at a density of 100 mm, and complete culture medium (high-sugar DMEM+20% FBS) was added. Change the medium the next day, add complete cell culture medium with a final concentration of zeocin of 300ng / mL to continue the culture, and change the medium every 2 days. After 7-10 days, observe the cell clones under a microscope, and mark more than 100 cell clones. Under sterile conditions, place a plastic...

Embodiment 2

[0055] Example 2 Obtaining Transgene-Positive Somatic Cell Nuclear Transfer Blastocysts

[0056] The fresh pig ovary taken from the slaughterhouse is washed with sterilized physiological saline, the follicle fluid is extracted from the follicle with a syringe, added to a sterile 10cm petri dish, and the eggs are picked under a stereomicroscope with a suction pipe and put into the pig's ovary. Wash 3 times in oocyte maturation solution, transfer to a four-well plate containing 700 μl of maturation solution per well, cover with 300 μl of paraffin oil, and place in 38.5°C, 5% CO 2 Cultivate in the incubator for 42-44h. After 42-44 hours, the oocytes expelled from the first polar body were selected, and the transgene-positive pig fetal fibroblasts prepared in Example 1 were prepared for somatic cell nuclear transfer (SCNT). Transfer the fused reconstituted embryos to each well of a four-well plate containing 700 μl of porcine oocyte development solution and covered with 300 μl of...

Embodiment 3

[0057] Example 3 Preparation of feeder layer MEF cells

[0058] Inoculate the 3rd generation mouse embryonic fibroblasts (MEF cells) into a 10cm cell culture dish at 38.5°C in 5% CO 2 When cultured to a confluence of 95%, add 10 μg / ml mitomycin-C to the DMEM culture medium containing 10% serum (FBS) for 2.5 hours; wash 4 times with PBS; routinely digest, and then count the cells; press 1 ×10 5 piece / cm 2 The density was spread on a four-well plate pre-treated with gelatin, and then placed at 38.5 ° C, 5% CO 2 Incubate overnight in an incubator.

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 provides a method for constructing a porcine naive embryonic stem cell line, and belongs to the technical field of cell biology. The method combines iPSCs technology with traditional embryonic stem cell technology to first establish a transgenic positive porcine fetal fibroblast cell line that simultaneously expresses mOCT4, mSOX2, mKLF4 and mc-MYC; obtaining a transgenic positive porcine blastocyst; and inoculating an inner cell mass of the transgenic positive porcine blastocyst to a cell feeder layer, starting DOX-induced exogenous transcription factor expression to obtain a porcine embryonic stem cell line; after the porcine embryonic stem cell line is stable in passage, stopping the expression of the exogenous mOCT4, mSOX2, mKLF4 and mc-MYC, and maintaining the stemness of stem cells by activated endogenous pOCT4, pSOX2, pKLF4 and pc-MYC to obtain the porcine naive embryonic stem cell line. The stem cells for genetic modification and long-term culture are provided forstudy of transgenic cloned pigs.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to methods for culturing and establishing lines of pig naive embryonic stem cells. Background technique [0002] Naive embryonic stem cells (ESCs) are true totipotent stem cells derived from the inner cell mass (ICM) of the blastocyst, with unlimited self-renewal and differentiation capabilities (Nichols J, 2009, Cell stem cell4(6) :487-492), which has extremely important application value in the research aspects of cell differentiation, development regulation, gene modification and regenerative medicine. The traditional embryonic stem cell line establishment method is to inoculate the inner cell mass of the pre-implantation blastocyst in a specific stem cell solution for culture, so as to obtain stem cell clones. At present, following mice, rat naive embryonic stem cells have been successfully established by adjusting the culture system (Li P, 2008, Cell 135(7):1299-1310). T...

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): C12N5/10A01K67/027
CPCA01K67/0278A01K2217/05A01K2227/108C12N5/0606C12N2500/32C12N2500/38C12N2500/44C12N2501/119C12N2501/235C12N2501/33C12N2501/405C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2501/727C12N2502/02
Inventor 李荣凤张曼玲姜海滨杨宁
Owner NANJING MEDICAL 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