Culture medium for rat embryonic stem cells

An embryonic stem cell and culture medium technology is applied in the culture medium field of rat embryonic stem cells, which can solve the problem of unstable karyotype of rat stem cells, and achieve the effect of ensuring karyotype and inhibiting differentiation.

Inactive Publication Date: 2013-04-10
谌兵来
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the karyotype of the rat stem cells is still very unstable after drug and gene targeting, and a stable rat stem cell line for injection has to be re-screened

Method used

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  • Culture medium for rat embryonic stem cells
  • Culture medium for rat embryonic stem cells
  • Culture medium for rat embryonic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Medium preparation

[0159] Add 10mM CHIR99021, 10mMPD0325901 and 10mM Pluripotin (SC1) to 100ml N2B27 mixed medium in sequence, so that the final concentration of CHIR99021 is 3μmol / L; the final concentration of PD0325901 is 1μmol / L; the final concentration of Pluripotin (SC1) is 1μmol / L The preferred culture medium of the present invention can be stored at 4°C for 1 month.

[0160] Among them, the N2B27 mixed medium is prepared according to the following formula:

[0161] DMEN / F12-N12 medium 100ml;

[0162] Neural cell basal medium / B27 medium 100ml;

[0163] 0.1M β-mercaptoethanol 200μl.

[0164] DMEN / F12-N12 medium is prepared according to the following formula:

[0165] DMEN / F12 medium 100ml;

[0166] N2 additive 1ml.

[0167] Neural cell basal medium / B27 medium is prepared according to the following formula:

[0168] Neural cell basal medium 100ml;

[0169] Nerve cell culture supplement factor B27 2ml;

[0170] 200mM L-glutamine 0.5ml.

Embodiment 2

[0172] Medium preparation

[0173] 10mM CHIR99021 and 10mM PD0325901 were added to 100ml DMEM medium successively, so that the final concentration of CHIR99021 was 3μM; the final concentration of PD0325901 was 1μM to obtain rat stem cell culture medium, which can be stored at 4℃ for 1 month.

[0174] Among them, the N2B27 mixed medium is prepared according to the following formula:

[0175] DMEN / F12-N12 medium 100ml;

[0176] Neural cell basal medium / B27 medium 100ml;

[0177] 0.1M β-mercaptoethanol 200μl.

[0178] DMEN / F12-N12 medium is prepared according to the following formula:

[0179] DMEN / F12 medium 100ml;

[0180] N2 additive 1ml.

[0181] Neural cell basal medium / B27 medium is prepared according to the following formula:

[0182] Neural cell basal medium 100ml;

[0183] Nerve cell culture supplement factor B27 2ml;

[0184] 200mM L-glutamine 0.5ml.

Embodiment 3

[0186] Primary cultured rat embryonic stem cells

[0187] Fibroblasts treated with MMC lose their mitotic activity and are planted in a 96-well plate to form a feeder layer.

[0188] The feeder layer prepared by the above method is used in 1-3. Aspirate the supernatant from the 96-well plate, and add the medium prepared in Example 1, 150 μl per well.

[0189] Use the oral control tube to suck the white rat blastocyst on day 4.5, its shape is like figure 2 (A) and figure 2 As shown in (B), add one blastocyst to each hole and place it at 37℃, 5% CO 2 And 5% O 2 Inside the incubator. The first day of planting looks like figure 2 (C) Shown. Incubate for 3 days without changing the medium.

[0190] Cultivate to the 4th day, such as figure 2 As shown in (D), the blastocyst is fixed on the feeder layer to form a larger cell cluster. The central part of the cell cluster was mechanically picked and transferred to another hole containing feeder layer, which was recorded as the first gene...

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PUM

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Abstract

The invention discloses a culture medium for rat embryonic stem cells. The culture medium comprises basal culture media and additives, wherein the additives comprise differentiation inhibitors and Pluripotin; and moreover, the differentiation inhibitors comprise GSK (Glycogen Synthase Kinase) inhibitors, FGFR (Fibroblast Growth Factor Receptor) inhibitors and MAPK (Mitogen-activated Protein Kinases) inhibitors. The invention also discloses a culture medium kit of the rat embryonic stem cells, a method for culturing the rat embryonic stem cells and applications of the culture medium and the kit, wherein the culture medium kit comprises the basal culture media, the differentiation inhibitors and the Pluripotin. With the adoption of the differentiation inhibitors and the Pluripotin, the culture medium for the rat embryonic stem cells can inhibit the differentiation of the rat embryonic stem cells and can further guarantee the stability of karyotype for reproductive potential.

Description

Technical field [0001] The invention relates to the field of cell culture. More specifically, the present invention relates to a culture medium for culturing rat embryonic stem cells and a corresponding culturing method. Background technique [0002] Gene sequencing of humans, mice, rats, and many animals and plants has been completed, marking that life science research has begun to enter the post-genome era, that is, the era of gene function analysis. The current gene function analysis methods include: gene knockout, antisense technology, dominant negative, gene induced overexpression, RNA interference, and bioinformatics, among which the gene knockout method is the most standard method for gene function analysis. Knockout analysis has become the gold standard for gene function analysis. [0003] Model organisms (including knockout rats and mice) will play an irreplaceable role in functional genomics research. In foreign countries, mice, rats, fruit flies, nematodes, and Xenopu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
Inventor 谌兵来曾桥刘上峰徐俊
Owner 谌兵来
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