Method for obtaining mouse haploid stem cells

A technology of stem cells and haploids, applied in the biological field

Active Publication Date: 2020-11-03
NANKAI UNIV
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  • Claims
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Problems solved by technology

However, there is no report on the establishment of mouse haploid epiblast stem cell lines (haEpiSCs) after implantation embryos. due to state

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  • Method for obtaining mouse haploid stem cells
  • Method for obtaining mouse haploid stem cells
  • Method for obtaining mouse haploid stem cells

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Embodiment Construction

[0025] In this study, we microinjected p53-knockout (p53-KO) mouse haESCs into wild-type mouse blastocysts, and transplanted the reconstituted embryos back into 0.5-day-old pseudopregnant mother mice In the oviduct of the embryo, the dissection was performed when the embryo developed to E6.5 days ( figure 1 ), isolate the Epiblast (epiblast) region and plant it in the culture medium, and finally form the standard mouse epiblast stem cells ( figure 2 ). Using the wild-type diploid mouse epiblast stem cell line as a control, we expanded and sorted the established haEpiSCs line by flow cytometry (FACS) ( image 3 ).

[0026] Material method:

[0027] 1. Obtaining of reconstructed embryos:

[0028] 1) Perform superovulation treatment on CD1-background female mice (48 hours after intraperitoneal injection of PMSG hormone, intraperitoneal injection of hCG hormone again), and then put superovulated female mice and male mice into cages, and leave the cages for later. The dams we...

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Abstract

The invention discloses a method for obtaining mouse haploid stem cells. The method comprises the following steps of: 1, injecting p53 knocked-out mouse haploid embryonic stem cells into a wild type mouse blastocyst by a microinjection method to obtain a reconstructed embryo; step 2, transplanting the reconstructed embryo back to an oviduct of a 0.5-day false pregnant female mouse or a uterus of a2.5-day false pregnant female mouse, performing dissecting when the embryo develops to E6.5 days, and separating an epiblast area; and step 3, culturing the epiblast region in a culture medium to finally form the mouse haploid epiblast stem cells. According to the invention, through a p53-KO method, spontaneous doubling of haploid stem cells in an in-vivo differentiation process is effectively inhibited, and a mouse haploid epiblast stem cell line derived from implanted epiblast tissue is successfully established in vitro by a chimeric technology.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for obtaining mouse haploid stem cells. Background technique [0002] Mammalian haploid cell lines have attracted considerable attention over the past decade due to their advantage in generating homozygous genotypes and are therefore widely used in forward and reverse genetic screens. Haploid embryonic stem cells (haESCs) are haploid pluripotent stem cells derived from pre-implantation haploid blastocysts. Because of their homozygous and stem cell advantages, they have shown great advantages in gene function screening and other aspects. However, haESCs, as undifferentiated pluripotent stem cells, have limitations in some lineage-specific genetic screens. [0003] Mouse epiblast stem cells (EpiSCs) are derived from post-implantation epiblast tissue (Epiblast), which are very similar to human embryonic stem cells (ESCs) in terms of morphology and various molecular biological ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2509/00
Inventor 高倩帅领张文豪赵乙丁
Owner NANKAI UNIV
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