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Orphan and haploid stem cell line and its preparation method and application

A haploid cell, solitary technique, used in applications, embryonic cells, animal cells, etc.

Active Publication Date: 2015-09-30
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no haploid cell that can replace sperm

Method used

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  • Orphan and haploid stem cell line and its preparation method and application
  • Orphan and haploid stem cell line and its preparation method and application
  • Orphan and haploid stem cell line and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 Establishment of androgenic haploid stem cells

[0114] Preparation of male haploid embryos:

[0115] Sperm: References (Kimura and Yanagimachi, 1995; Yang et al., 2011), collected sperm from Oct4-EGFP transgenic mice (purchased from Nanjing Institute of Model Animals) for intracytoplasmic sperm injection (ICSI) Prepare.

[0116] Egg cells: egg cell donors were from B6D2F1 (C57BL / 6×DBA2) female mice, and B6D2F1 female mice were purchased from Nanjing Institute of Model Animals.

[0117] Male haploid embryos were prepared using the following method:

[0118] The first method, the standard nuclear transfer method (ref. n et al., 2011; Wakayama et al., 1998), in which the somatic cell donor is replaced by the sperm head. Specifically, the oocytes were obtained 14 hours after the mice were injected with chorionic gonadotropin (HCG), and then cultured in HEPES-CZB medium containing 5 μg / ml cytochalasin B; It is customary to perform enucleation; after enucleatio...

Embodiment 2A

[0123] Pluripotency of Example 2AG-haESCs

[0124] AG-haESCs have the same clonal morphology as normal diploid mouse embryonic stem cells. Immunofluorescence analysis showed that markers of ES cells (including Nanog, Oct4, Sox2, and Ssea1) were present in haES clones ( figure 2 A) and in cells with only a single DNA content obtained by FACS (Table 1).

[0125] Table 1

[0126]

[0127] Gene expression profiles of normal ES cells and embryonic fibroblasts (MEFs) from male individuals were compared with AG-haESCs. To avoid the influence of diploidized cells on expression profiles, samples in G1 / G0 phase were collected by FACS. Cluster analysis of these cells obtained from microarray expression data showed that AG-haESCs had a high similarity to diploid ES cells, but not to MEFs ( figure 2 B).

[0128] To test the developmental potential of AG-haESCs, Actin-EGFP-labeled haploid cells (AGH-EG-1) were injected into ICR-derived diploid blastocysts to obtain chimeric mice wi...

Embodiment 3

[0130] Example 3 Partial Maintenance of AG-haESCs Paternal Genome Imprint

[0131] Since the paternal imprint established at the stage of primordial germ cells prevails throughout the fertilization process and continues throughout the embryonic development process. In this example, the inventors tested whether these AG-haESCs maintained paternal imprinting.

[0132] Firstly, the expression of imprinted genes was compared between haploid and control diploid ESCs, and it was found that all paternal genes of AG-haESCs derived from androgens were imprinted, and genes expressed by maternal sources were down-regulated, except for the H19 gene; on the contrary, Maternally imprinted genes (expressed on paternal alleles) were upregulated (Table 1 and image 3 A). It indicated that AG-haESCs largely maintained a typical paternal imprinting background.

[0133] In order to further evaluate its epigenetic status, the methylation background of two paternally imprinted genes Gtl2 and H19...

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Abstract

Provided are an androgenetic haploid stem cell line, preparation method and use thereof. Specifically, provided are an androgenetic haploid cell and androgenetic blastula. The nucleus of the cell or the blastula only comprises a haploid autosome and a sex chromosome, the sex chromosome being the X chromosome and containing no Y chromosome. The androgenetic haploid cell of the present application can replace a spermatid as a ligand to generate a fertile animal individual, thus facilitating gene manipulation and being capable of transferring genetic information to the offspring.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to an androgenic haploid stem cell line and its preparation method and application. Background technique [0002] All mammals are diploid, that is, there are two sets of chromosomes in the cell, one from the father and one from the mother. In sexually reproducing individuals, haploid gametes (eggs and sperm) can mediate gene transmission to the next generation. However, sperm cannot be cultured in vitro, which greatly limits its genetic manipulation; Therefore, diploid cells are a huge difficulty for gene research. [0003] Haploid cells are convenient for genetic research because they contain only one set of chromosomes. Although mouse haploid embryos have been obtained at present, the embryonic stem cells obtained from these haploid embryos will later show a diploid karyotype. Parthenogenetic mouse haploid embryonic stem cell lines (haESCs) can be established...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735C12N5/073C12N5/10C12N15/85C12N15/06A01K67/027
CPCC12N5/0603
Inventor 李劲松杨辉施霖宇钟翠青梁丹
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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