A method for obtaining mouse haploid stem cells

A haploid, stem cell technology, applied in the biological field

Active Publication Date: 2022-05-17
NANKAI UNIV
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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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  • A method for obtaining mouse haploid stem cells
  • A method for obtaining mouse haploid stem cells
  • A 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, comprising the following steps: step 1, injecting p53-knockout mouse haploid embryonic stem cells into wild-type mouse blastocysts by microinjection Obtain reconstructed embryos; step 2, transfer the reconstructed embryos back to the fallopian tube of a 0.5-day pseudopregnant mother mouse or the uterus of a 2.5-day pseudopregnant mother mouse, dissect the embryo when it reaches E6.5 days, and separate the epiblast region Step 3, culturing the epiblast region in the culture medium to finally form mouse haploid epiblast stem cells. The present invention passes p53 The ‑KO method effectively inhibited the spontaneous diploidization of haploid stem cells during differentiation in vivo, and successfully established mouse haploid epiblast stem cells derived from post-implantation epiblast tissue in vitro by means of chimera technology Tie.

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 Patents(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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