Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Somatic cell nuclear transplantation method and application thereof

A technology of somatic cells and embryonic stem cells, applied in botany equipment and methods, applications, embryonic cells, etc., can solve the problems of cloning mammalian defects that have little effect and exceed the ability of reprogramming, and achieve the effect of improving cloning efficiency

Pending Publication Date: 2021-12-17
INST OF ZOOLOGY CHINESE ACAD OF SCI
View PDF0 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Progress has been made in identifying and overcoming key epigenetic barriers to nuclear transfer, including inhibition of histone deacetylation, reduction of DNA methylation, and removal of somatic heterogeneity by H3K9me3 (Dai et al., 2010; Gao et al; 2018; Kishigami et al, 2006; Matoba et al., 2014), these methods can significantly improve the developmental efficiency of nuclear transfer embryos, but have little effect on the defects of all cloned mammals
Although imprinting abnormalities have also been observed in cloned animals, typical genomic imprinting mediated by DNA methylation is relatively stable beyond the current capacity of oocyte cytoplasmic reprogramming (Humpherys et al., 2014)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Somatic cell nuclear transplantation method and application thereof
  • Somatic cell nuclear transplantation method and application thereof
  • Somatic cell nuclear transplantation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0240] 11. RNA-Seq library preparation and data analysis

[0241] Mince the placenta and digest it with 0.25% trypsin at 37°C. During the digestion process, blow and suck several times with the tip of a pipette to make the digestion more comprehensive, and stop the digestion after 10-15 minutes. GFP-positive placental cells were sorted by flow cytometry and analyzed by RNA sequencing after collection.

[0242] For transcriptome analysis in mice, brain tissues from WT mice, Δ4-NT-3KO and native mice were collected for RNA-Seq. Total RNA was extracted from fetuses using TRIzol, followed by reverse transcription-polymerase reactions with 1 μg of purified RNA each time.

[0243] RNA-seq library construction and data analysis: 2 rounds of PolyA-tailed RNA purification were performed for each sample. 150bp paired-end sequencing was performed with an Illumina HiSeq4000 sequencer. RNA-seq data were analyzed with HISAT2 (version 2.1.0) and Cufflinks (version 2.2.1). Data statistics...

Embodiment 1

[0254] Example 1 Abnormal biallelic expression of H3K27me3 imprinted gene in E19.5 clone placenta

[0255] In order to study the specific expression of the H3K27me3 imprinted gene, the offspring of two inbred strains of mice were used as the research object in the present invention.

[0256] experimental method:

[0257] The female parent is a female mouse of the C57BL / 6-Tg(CAG-EGFP)1Osb / J strain, and the male parent is a male mouse of the PWK / PhJ strain. Somatic cell nuclear transfer was performed using granulosa cells from hybridized F1 mice, and the specific steps were as described in Experimental Method 7 above (that is, the part of somatic cell nuclear transfer and embryo culture). At the same time, C57 (GFP+) and PWK sperm and oocytes were used as controls for in vitro fertilization. These imprinted genes were not detected after E9.5. The inventors therefore analyzed E10.5 SCNT and IVF embryos. Fetal-derived placental cells with green fluorescence are isolated by flo...

Embodiment 2

[0267] Example 2 Monoallelic knockout of four H3K27me3 imprinted genes can significantly improve cloning efficiency

[0268] experimental method:

[0269] The invention discloses a method for improving the cloning efficiency of mice, a cell line and a model mouse which can be used for improving the cloning efficiency. The specific steps are:

[0270] 1. Obtain oocytes. Select female mice of appropriate age, inject pregnant horse serotonin (PMSG), and inject human chorionic gonadotropin (HCG) within 13-17 hours after injection. The egg retrieval operation was performed 13-15 hours after the injection of HCG. The collected oocytes were washed with Hepes-CZB, then cultured in M16 (Sigma) medium, and placed in a 37°C, 5% CO2 incubator for further use. Granulosa cells were removed by digestion with hyaluronidase, and oocytes were temporarily stored in CZB medium containing 3 mg / mL BSA prior to micromanipulation.

[0271] 2. Obtain haploid embryonic stem cells in which Sfmbt2, ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a somatic cell nuclear transplantation method and application thereof. According to the method, a single allele is obtained by using a haploid embryonic stem cell system, and meanwhile, one or more donor somatic cells in 26 H3K27me3 related imprinting genes are knocked out for nuclear transplantation. Results show that a single allelic knockout of one or more of Slc38a2, Slc38a4, Sfmbt2, Etv6, Platr4, Gramd1b, Slc38a1, Gab1, Mbnl2, Smoc1, Bmp7, Rbms1, Fam198b, Sh3gl3, Hunk, Jade1, E2f3, Tle3, Runx1, Epas1, Bbx, Enc1, Inhbb, Sox21, Otx2, Rbp2 (e.g., one or more of four of Sfmbt2, Jade1, Gab1 and Smoc1)of the H3K27me3 related imprinting genes, the imprinting expression pattern of the genes tends to be normal. Meanwhile, the somatic cell cloning efficiency can be improved to 14% when the donor somatic cells are used for cloning, and the somatic cell cloning efficiency of a wild type control group is 0. In addition, according to the cloned animal obtained by the method, a large placenta-large fetus phenomenon is corrected.

Description

technical field [0001] The invention relates to the field of animal biotechnology, in particular, the invention relates to a somatic cell nuclear transfer method and its application. Background technique [0002] Somatic cell nuclear transfer (SCNT) can reprogram somatic cell nuclei to a totipotent state, which has great application potential in the fields of animal breeding and regenerative medicine (Rideout et al., 2001). However, the extremely low developmental efficiency and frequently observed developmental abnormalities of nuclear transfer embryos suggest an epigenetic barrier to somatic cell reprogramming (Lu and Zhang, 2015). Of these abnormalities, large fetus syndrome (LOS) is the most common and has been found in cloned cows, sheep and mice. The syndrome refers to a heterogeneous group of symptoms including excessive birth size and severe birth defects (Yang et al., 2007). Progress has been made in identifying and overcoming key epigenetic barriers to nuclear tr...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N5/10C12N15/113C12N15/877A61K35/545A61K35/33A61P43/00A61P3/10A61P15/00A61P3/04A01K67/027
CPCC12N5/0606C12N5/0603C12N5/0656C12N15/113C12N15/877A61K35/545A61K35/33A61P43/00A61P3/10A61P15/00A61P3/04A01K67/0276C12N2310/20A01K2207/15A01K2217/075A01K2227/105A01K2227/101A01K2227/108A01K2227/106A01K2227/102A01K2227/103A01K2267/03
Inventor 周琪李伟王乐韵李治琨王立宾
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com