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Method for constructing chimeric rat using rat embryonic stem cells

A technology of pluripotent stem cells and embryos, applied to embryonic cells, germ cells, animal cells, etc., can solve the problems of blocking human ES cells and not reporting the usefulness of ROCK inhibitors

Inactive Publication Date: 2012-10-31
NAT CANCER CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In recent years, Watanabe et al. have found that the Rho-binding kinase (Rho-associated kinase; ROCK) inhibitor Y-27632 blocks apoptosis in human ES cells and induces growth after isolation of single cells by enzymatic treatment (non- Patent Document 5), and there is no report on the usefulness of ROCK inhibitors for human stem cell culture (Patent Document 2)

Method used

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  • Method for constructing chimeric rat using rat embryonic stem cells
  • Method for constructing chimeric rat using rat embryonic stem cells
  • Method for constructing chimeric rat using rat embryonic stem cells

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preparation example Construction

[0096] The culture in the above production method is preferably performed using a medium containing at least two ES cell differentiation inhibitors, more preferably using a medium containing at least two ES cell differentiation inhibitors and a ROCK inhibitor. Here, "ES cell differentiation inhibitor" refers to a substance other than leukemia inhibitory factor (LIF) that can inhibit the differentiation of ES cells into other cells or tissues, etc., and in the present invention, may be a substance that inhibits Any substance that acts on the differentiation of ES cells. ES cell differentiation inhibitors include, for example, MEK inhibitors, GSK3 inhibitors, TGFβ receptor inhibitors, FGF receptor inhibitors, and the like. For the cultivation, specifically, a medium containing a MEK inhibitor and a GSK3 inhibitor is preferably used; more preferably a medium containing a MEK inhibitor, a GSK3 inhibitor, an FGF receptor inhibitor, and a ROCK inhibitor is used; It is more preferab...

Embodiment 1

[0194] Example 1: Preparation of fluorescent reporter gene transgenic rats

[0195] To establish and validate ES cells, transgenic rats were prepared for monitoring the expression of the Oct4 gene by fluorescence. Oct4 promoter region DNA was amplified from Wistar rat genomic DNA by PCR using KOD Ver.2 DNA polymerase (Toyobo), and inserted into pCS2-Venus plasmid. The Oct4 promoter-Venus DNA was injected into the pronuclei of fertilized eggs of Wistar rats to obtain Oct4-VENUS transgenic rats.

Embodiment 2

[0196] Embodiment 2: Preparation of rat ES cells

[0197] (1) Preparation of reagents and feeder cells

[0198] First, 4 inhibitors were prepared, namely, Y -27632 as a ROCK inhibitor (WAKO Company), P D0325901 as a MEK inhibitor (Axon Medchem Company), A -83-01 as a type I TGF-β receptor inhibitor (TOCRIS Company) and C HIR99021 acts as a GSK3 inhibitor (Axon Medchem Company) (hereinafter, these 4 inhibitors are referred to as "YPAC factors").

[0199] For ES cells, 20% FBS (Lot No. 1204059: GIBCO Company), 0.1 mM 2-mercaptoethanol (Sigma Company), 1% non-essential amino acid stock solution (GIBCO Company), and 1% 1× antibiotic antimycotic ( GIBCO Company) was added to DMEM (GIBCO Company) containing 110 mg / L sodium pyruvate and 200 mM GlutaMax to prepare the basal medium for ES cells.

[0200] YPAC factors were added to this basal medium for ES cells to give the following concentrations: 10 μM ROCK inhibitor Y-27632, 10 μM MEK inhibitor PD0325901, 0.5 μM type I TGF-β ...

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Abstract

Provided is a method for constructing a chimeric embryo and a chimeric rat, characterized by comprising contacting rat pluripotent stem cells with a host embryo in the presence of an ES cell differentiation inhibitor. This method comprises a step (a) for contacting a host embryo, which is collected from a female rat after fertilization, with rat pluripotent stem cells in the presence of an ES cell differentiation inhibitor; and a step (b) for incubating the host embryo having been contacted with the rat pluripotent stem cells to form a chimeric embryo.

Description

technical field [0001] The present invention relates to a method for preparing chimeric embryos with improved germline transmission efficiency using rat pluripotent stem cells, particularly rat embryonic stem cells (hereinafter referred to as "ES cells"), using the chimeric A method for preparing a chimeric rat from an embryo, a chimeric rat prepared by the method, and a medium useful for preparing the chimeric rat. Background technique [0002] ES cells are an almost totipotent cell line, very suitable for the preparation of genetically modified animals and the like. For example, chimeric animals can be produced by injecting ES cells (in which specific genes have been disrupted) into normal host blastocysts to be mixed with cells from the host embryo, and returning the mixture to the uterus, by crossing the resulting chimeric animals or offspring, and selecting born animals, can produce genetically modified animals (knockout animals) in which specific genes have been disru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073A01K67/02A01K67/027C12N5/0735
CPCA01K67/0271A01K2267/0393C07K2319/81A01K2207/20A01K2217/052A01K2227/105A01K67/0275A01K2217/05C12N9/22A01K2217/00C12N15/8509A01K2227/10A01K67/0276C12N15/873C12N5/0606C12N15/85C12N2500/30C12N2501/065C12N2501/15C12N2501/998C12N2501/999
Inventor 落谷孝广川又理树
Owner NAT CANCER CENT
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