Use of protein kinase inhibitor for inhibiting diploidization of haploid cells

A technology of protein kinase inhibitors and haploid cells, applied in the field of haplotype maintenance in in vitro culture or differentiation of haploid cells, can solve the problem of failure to effectively control the diploidization of haploid embryonic stem cells, inability to effectively Obtain haploid cell genetic screening and other issues to achieve the effect of simplifying the culture program and reducing costs

Active Publication Date: 2018-12-14
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In conclusion, it is still not possible to effectively control the diploidization of haploid embryonic stem cells during culture and differentiation (especially for rodent haploid somatic cells), nor to efficiently obtain haploid cells for genetic selection

Method used

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  • Use of protein kinase inhibitor for inhibiting diploidization of haploid cells
  • Use of protein kinase inhibitor for inhibiting diploidization of haploid cells
  • Use of protein kinase inhibitor for inhibiting diploidization of haploid cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Dynamic changes of chromosomes during the division of haploid embryonic stem cells

[0050] Diploid embryonic stem cell lines and orphan (parthenogenetic) haploid embryonic stem cell lines derived from fertilized eggs were established from the same strain of H2B-GFP transgenic mice (introduced from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.).

[0051] Diploid embryonic stem cell lines and orphan androgenic haploid embryonic stem cells were cultured in a 12-well plate. First, mouse feeder cells (E13.5-day embryonic fibroblasts of mice treated with mitomycin C (sigma, M0503)) were inoculated into culture well plates, and fibroblast culture medium (high glucose DMEM (Gibco, C12430500BT), 1× sodium pyruvate (100×, Gibco, 11360-070), 1× non-essential amino acids (100×, Gibco, 11140-050), 1× Penicillin-Streptomycin (Gibco, 15140163, 100× ) Add 10% fetal bovine serum (Gibco, 16000-044) to culture for use. Orphan androgenic (parthenogenetic) haploid e...

Embodiment 2

[0053] Example 2 Screening of Protein Kinase Inhibitors and Their Concentrations for Inhibiting Diploidization of Haploid Cells

[0054] (1) Screening of protein kinase inhibitors and their concentrations in 12-well plates.

[0055] First, the mouse feeder cells (mice MEF treated with mitomycin C (sigma, M0503)) were inoculated on a culture well plate, and fibroblast culture medium (same as Example 1) was added with 10% fetal bovine serum (Gibco, 16000-044) Cultivation for use. Particulate (parthenogenetic) haploid embryonic stem cells were stained with live cell dye 2μg / ml Hoechst 33342 (Invitrogen, H3570) at 37 degrees Celsius for 15-20 minutes, 1n peaks were collected by flow sorting, and diploid ES was used as a control 2n peak position. The enriched haploid embryonic stem cells were averagely planted on a 12-well plate with feeder cells. Using mouse embryonic stem cell culture medium (same as Example 1), add a specific concentration and specific type of protein kinase inhi...

Embodiment 3

[0063] Example 3 Using Rock kinase inhibitor to obtain haploid neural stem cells from haploid embryonic stem cells

[0064] Obtain flow-purified orphan androgenic (parthenogenetic) haploid embryonic stem cells, plant 50,000 cells in a 3.5 cm petri dish with feeder cells, and grow clones in 3-5 days, 0.25% trypsin (Gibco, 25200 -072) Digest for 3-5 min, stop the digestion with 10% FBS, wash once with 1×PBS, and use mouse embryonic stem cell culture medium without PD0325901, Chir99021 and LIF (same as Example 1) for suspension culture of haploid ES, suspension culture Two days later, 500 embryoid spheres were seeded on a 6 cm petri dish covered with polylysine 1mg / ml (sigma, P6407) and 5ug / ml laminin (invitrogen, 23017-015). After 16 hours, change to neural stem cell culture medium (N2B27 basic medium (same as Example 1), add 20ng / ml bFGF (R&D, 233-FB-001MG) and 20ng / ml EGF (R&D, 2028-EG-200)) . Add any Rock inhibitor such as 20μM Y-27632 and the same dose of DMSO as a control. ...

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Abstract

The invention provides the use of a CDK1 protein kinase inhibitor and/or a Rock protein kinase inhibitor for inhibiting the diploidization of haploid cells. The screened CDK1 protein kinase inhibitorand Rock protein kinase inhibitor can be utilized to widely culture and differentiate patrogenesis and parthenogenesis haploid embryonic stem cells. A technical scheme of the invention provides a basis for research and application based on haploid genome.

Description

Technical field [0001] The present invention relates to the field of cell culture. More specifically, the present invention relates to a method for maintaining haplotypes in haploid cells cultured or differentiated in vitro. Background technique [0002] For diploid organisms, haploids have a single-copy genome, which has great advantages in genetic research. However, haploid embryonic stem cells will be spontaneously doubled during in vitro culture or differentiation. To obtain or maintain haploid embryonic stem cells, continuous flow enrichment is required, which greatly increases the packing of haploids. Cost, thus hindering its application in genetic screening and modification (Elling et al., 2011; Leeb and Wutz, 2011; Yang et al., 2012; Zhou et al., 2016); at the same time, the maintenance of the ploidy of haploid stem cells Mechanism is also an interesting question, which may contain the unique cell cycle regulation mechanism of embryonic stem cells. [0003] In recent year...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735C12N5/077C12N5/0793C12N5/0797
CPCC12N5/0606C12N5/0619C12N5/0623C12N5/0657C12N2501/727C12N2506/02C12N2506/08
Inventor 周琪李伟何正泉王昱凯夏宝龙
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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