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Induction and culture line establishment method for mammal parthenogenetic epiblast stem cells and obtained parthenogenetic epiblast stem cell line

A mammalian and stem cell technology, applied in the field of mammalian reproductive and developmental bioengineering, can solve the problem of not obtaining epiblast-like stem cells, etc.

Inactive Publication Date: 2017-12-29
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, with the development of animal research in life sciences, methods for inducing mammalian stem cells have emerged in an endless stream, and the types of stem cells have become more subdivided. However, no epiblast-like stem cells (EpiSCs like cell line)

Method used

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  • Induction and culture line establishment method for mammal parthenogenetic epiblast stem cells and obtained parthenogenetic epiblast stem cell line
  • Induction and culture line establishment method for mammal parthenogenetic epiblast stem cells and obtained parthenogenetic epiblast stem cell line
  • Induction and culture line establishment method for mammal parthenogenetic epiblast stem cells and obtained parthenogenetic epiblast stem cell line

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1, the preparation of mouse early embryo

[0092] 1. Perform superovulation on 8-week-old mice, select female mice of ICR and 129 strains for intraperitoneal injection of PMSG, and after 48 hours, intraperitoneal injection of HCG, (injection volume: 7.5IU (international unit) / only; injection The time is 17:00-19:00), after 16 hours, eggs were collected, and unfertilized oocytes were recovered from the ampulla of the oviduct of the mouse, and used for the following stem cell induction and culture establishment. Move the oocytes without granulosa cells to the M2 droplet and wash them three times, put them in KSOM to balance for 30-60min, and move them to the activation solution containing strontium chloride + cytochalasin B for parthenogenetic activation.

[0093] 2. Parthenogenesis

[0094] After being activated for 5 h, the oocytes were washed three times in M2 droplets, and then transferred to KSOM for culture (37°C, 5% CO 2 ), the pronucleus of the oocyte...

Embodiment 2

[0097] Example 2, mouse pa-EpiLSCs pluripotent stem cell induced line establishment and culture medium composition

[0098] The superovulated mouse oocytes recovered by the above method for 16 hours were activated for 5 hours in the activation solution containing strontium chloride + cytochalasin B, and then cultured in KSOM for 96 hours to blastocysts. , Acidic) to remove the zona pellucida of mouse embryos, and then add fibroblast growth factor 2, activin A, 5% The specific culture medium (AF medium) with defined serum substitutes was used to induce pluripotent stem cell lines. Replace the culture medium after 5 days: AF+2% serum replacement, primary culture for 8 days. Eight days later (P1), the clones should be picked for subculture, and the culture medium is AF+2% serum replacement. After 12 days (P2), the subculture method remains unchanged, and the culture medium is AF+1% serum substitute. After 14 days (P3), the subculture method remains unchanged, and the culture m...

Embodiment 3

[0102] Example 3. Analysis of mouse pa-EpiLSCs stem cell characteristics and developmental potential

[0103] 1. AP (alkaline phosphatase) staining

[0104] Preparation method of AP dyeing solution: ① Dyeing solution No. 1 Sodium nitrite solution (1:50) and dyeing solution No. 2 FRV-alkaline solution (1:50) are mixed according to 1:1, and room temperature is protected from light for 2-3 minutes; ② Dyeing solution 3naphthol-AS-BIalkaline solution (1:50), added to ultrapure water. Add the prepared dye solution ② into the dye solution ① and mix well. Store it away from light for later use, and prepare it now.

[0105] When pa-EpiLSCs were cultured on a 4-well plate until the second day, the culture medium was discarded, and 500 μL of 4% paraformaldehyde was added to fix at room temperature for 30 min. After discarding the paraformaldehyde, wash once with phosphate buffer, discard the phosphate buffer, add 500 μL of AP staining solution prepared in advance, and store overnight ...

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Abstract

The invention discloses an induction and culture line establishment method for mammal parthenogenetic epiblast stem cells and an obtained parthenogenetic epiblast stem cell line. The method comprises the following steps: executing the superovulation to two varieties of female mice ICR and 129, and executing the parthenogenetic activation to ovocyte; after 5 h of the activation, cultivating for 96 h in KSOM, and generating blastaea, and executing the line establishment by using the blastaea. The line establishment method is capable of efficiently inducing mice parthenogenetic embryo to form the parthenogenetic epiblast stem cells pa-EpiLSCs, keeping the parthenogenetic diploid features and steady growing for more than 30 generations. The method is capable of providing a new tool for rapid generation of a recessive trait genetic model, and providing the new technical and theoretical basis for other mammals establishing the pa-EpiLSCs from the parthenogenetic embryos.

Description

technical field [0001] The present invention relates to mammalian reproductive development bioengineering technology, in particular to a method for inducing and cultivating mammalian epiblast stem cells and the obtained parthenogenetic epiblast like stem cell (pa-EpiLSCs) . Background technique [0002] Embryonic stem cells (ESCs) can retain the ability to generate any type of cells in vivo indefinitely, and can proliferate infinitely in vitro and maintain undifferentiated abilities, so they not only have great application value for tissue repair and regeneration, but also for human diseases. It provides a powerful tool for modeling and studying life activities. In the 1990s, the research on animal embryonic stem cells (ES) had already started, and the establishment of mouse and human embryonic stem cell lines was isolated for the first time, so scientists won the 2009 Nobel Prize. In the past 20 years, the research on embryonic stem cells has been a hot spot in the life s...

Claims

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Application Information

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IPC IPC(8): C12N5/0735A01N1/02
CPCA01N1/0221C12N5/0606C12N2500/62
Inventor 包斯琴吴宝江
Owner INNER MONGOLIA UNIVERSITY
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