18 results about "Mouse Embryonic Stem Cell" patented technology

The invention provides an enhancer RNA (Ribonucleic Acid) molecule MZGAe1 and application thereof, and relates to the technical field of biology. The invention provides an enhancer RNA (Ribonucleic Acid) molecule MZGAe1. The nucleotide sequence of the enhancer RNA molecule MZGAe1 is as shown in SEQ ID NO: 1; meanwhile, the invention further provides a specific sgRNA sequence for activating the molecule in a targeted manner and a recombinant vector of the specific sgRNA sequence. Expression of endogenous MZGAe1 of cells is specifically activated by adopting a CRISPR activation technology, and conversion of mouse embryonic stem cells to bicellular-like cells can be efficiently promoted. By providing a brand-new endogenous targeted MZGAe1 accurate activation tool, the efficient transformation of wild mouse embryonic stem cells to bicellular cells can be realized only under the condition of endogenous activation of single enhancer RNA molecule MZGAe1, the proportion is at least increased by 5%, the expression of a totipotent marker gene ZSCAN4 is activated, and the expression of the totipotent marker gene ZSCAN4 is promoted. And an efficient and specific brand-new research tool and scheme are provided for researching zygote genome activation, embryonic development early events and cell reprogramming.

The invention relates to a method for inducing mouse trophoblast stem cells in vitro. According to the method, mouse embryonic stem cells are used as starting cells, Sorcs3 gene knockout mouse embryonic stem cells are obtained through a gene editing technology, and in-vitro induced trophoblast stem cells (iTSCs) are efficiently and rapidly obtained by means of a flow cytometry sorting technology and in combination with specific antibody sorting. According to the invention, the Sorcs3 gene is knocked out from the embryonic stem cell, so that the capability of the embryonic stem cell to differentiate to the trophoblast lineage can be obviously enhanced; an important research platform is provided for deeply researching a lineage intertransformation mechanism of cell masses (ICM) and trophoblast (TE) in the blastocysts, and the method has important significance for researching lineage limitation of early embryonic development of mammals and the like.

The invention discloses a vector system and method capable of converting mouse embryonic stem cells into embryonic-like cells in a 2-cell period, the vector system comprises a mouse Zfp352 gene sequence, and the nucleotide sequence of the mouse Zfp352 gene is shown as SEQ ID No.1. The invention also discloses a method for converting the mouse embryonic stem cells into embryonic-like cells in a 2-cell period. The invention creatively discovers that through overexpression of the Zfp352 in the mouse embryonic stem cells, the efficiency of converting the mouse pluripotent embryonic stem cells into the totipotent embryonic-like cells in the 2-cell period can be remarkably improved.

The application provides a non-coding RNA combination for inducing totipotency of mouse embryonic stem cells and application thereof, and relates to the technical field of biology.The combination comprises: an enhancer RNA with a sequence shown as SEQ ID NO:1 or SEQ ID NO:2, and a long-chain non-coding RNA with a sequence shown as SEQ ID NO:3.The application discloses, for the first time, that the CRISPR activation system is used to synergistically up-regulate the expression of the combination, and the conversion of mouse embryonic stem cells to two-cell-like cells can be efficiently promoted.The application does not need to integrate exogenous transcription factors, and thus the risk of genome is avoided; the sgRNA is precisely targeted, and thus the defect of non-specific off-target of chemical small molecules is overcome; a significant synergistic effect is generated, the induction efficiency of two-cell-like cells is significantly improved to about 11.6%, and a new strategy which is safer, more efficient and controllable is provided for stem cell fate reprogramming and early development research.

This invention belongs to the field of gene editing and epigenetics technology, and discloses a method based on dCasRx-FTO for m 6 A. Editing system and its recombinant expression plasmid, cell model and its application; this invention first constructs an m based on dCasRx-FTO. 6 The A-editing system, namely the dCasRx-FTO fusion protein, utilizes catalytically inactivated CasRx (dCasRx) as a highly specific RNA guidance scaffold to fuse with m 6 A demethylase FTO enables the targeting of a specific region m 6 Targeted removal of A-modification. Building upon this, the present invention further obtains monoclonal mouse embryonic stem cells (mESCs) stably expressing the dCasRx-FTO fusion protein. Combined with an sgRNA library targeting a specific set of transcripts, high-throughput screening can be performed on embryonic stem cells containing the 2C-like pluripotency reporter gene MERVL::tdTomato, thereby identifying key m-modification inhibitors suppressing the pluripotency state of mESCs at the transcriptome level. 6 A modification site, thus enabling its use to reveal m 6 The key role of A-modified targets in the regulation of stem cell pluripotency.

The application relates to a mouse model construction method for psoriasis research based on SBSN 600 lysine dihydroxyisobutyryl modification, which comprises the following steps: first step, constructing a plasmid comprising a DNA fragment of a SBSN gene sequence, wherein 600 sites of the SBSN sequence are mutated from lysine (K) to threonine (T); second step, culturing mouse embryonic stem cells; third step, electrically transforming the embryonic stem cells; fourth step, drug screening; fifth step, culturing the embryonic stem cells screened in the fourth step; sixth step, injecting the embryonic stem cells cultured in the fourth step into mouse blastocysts, then culturing the mouse blastocysts, and transplanting the cultured blastocysts into the uterus of a pseudopregnant female mouse, wherein the mouse delivered by the female mouse is a chimera mouse; and seventh step, breeding the chimera mouse and identifying the mouse genotype through PCR.

The present invention belongs to the field of animal genetic engineering and genetic modification. Specifically, it discloses a method for constructing a humanized epidermolysis bullosa DEB mouse model and its application in biomedicine. The method comprises the following steps: preparing genetically engineered mice using mouse embryonic stem cells, and replacing the full-length mouse Col7a1 gene sequence with a human COL7A1 genomic sequence or a mutant sequence thereof; preferably, the human COL7A1 genomic sequence is represented by hg19:chr3:48,635,720-48,603,572; the human COL7A1 genomic mutant sequence is represented by hg19:chr3:48,635,720-48,603,572&c.6527 dup C; the mutant animals constructed by the present invention can survive for 7-10 days, which is significantly longer than the median survival of 2 days reported for Col7a1- / - knockout mice, without drug maintenance, providing a longer window of survival for therapeutic experiments.

The application provides a non-coding RNA combination and application thereof in mouse totipotent stem cell induction, and relates to the technical field of biotechnology.The combination comprises: an enhancer RNA with a sequence as shown in SEQ ID NO:1 and SEQ ID NO:2, and a long-chain non-coding RNA with a sequence as shown in SEQ ID NO:3.The application discloses, for the first time, that the expression of the endogenous non-coding RNA combination is synergistically inhibited by using a CRISPR inhibition system, and the mouse embryonic stem cells can be efficiently promoted to transform into 2C-like cells.The application does not need to integrate an exogenous transcription factor, avoids the safety risk of the genome, overcomes the defect of non-specific off-target of a chemical small molecule by precise targeting, and significantly improves the induction efficiency of the 2C-like cells to about 9.09%.The application provides a safer, more efficient and controllable new strategy for stem cell fate reprogramming and early development research.

The invention provides a culture method for promoting conversion of mouse embryonic stem cells into totipotent stem cells. The cells can be kept in a brand new form and subcultured under the condition. According to the method adopted by the invention, an inhibitor WM-3825 of 2 mu M H4K16ac is added into a DMEM (Dulbecco Modified Eagle Medium) with a culture medium composition of 10% fetal calf serum according to a proportion of 1: 1000, the mouse embryonic stem cells can be normally cultured and passaged, the change of the totipotent gene is detected through real-time fluorescent quantitative PCR (Polymerase Chain Reaction), the expression of the totipotent related gene Dux is found to be obviously increased, and the expression of the mouse embryonic stem cells is obviously improved. Flow cytometry detection shows that the proportion of the totipotent stem cells is remarkably increased, and AP dyeing shows that the pluripotency of the mouse embryonic stem cells is reduced. The method starts from cell culture, and the method is possibly suitable for research on conversion from pluripotency to totipotency of embryonic stem cells of other mammals including human. By adding a small-molecule inhibitor WM-3825 of H4K16ac, a brand-new method for promoting conversion from the mouse pluripotent stem cells to the totipotent stem cells is established, and the 2-cell period of mouse embryonic development can be simulated in vitro through the culture system. The invention provides a new method for the generation of the totipotent stem cells, deepens the understanding of people on different states of the stem cells, and provides a new perspective for understanding an epigenetics mechanism between the conversion of the totipotent stem cells and the conversion of the pluripotent stem cells.

The invention relates to the technical field of cell culture, in particular to a method for induced culture of embryonic stem cells. The method comprises the following steps: resuscitating cryopreserved Balb / c mouse embryonic stem cells, culturing with a culture solution A containing leukemia inhibition factors, and continuously culturing with a culture solution B containing Activin A after passage; sequentially culturing in an HM culture medium added with specific cell factors, and cooperatively using a cell induction matrix prepared from various components such as hyaluronic acid, 3-aminophenylboronic acid, mono-ammonium glycyrrhizinate and the like. Through the synergistic effect of each culture step and each component in the cell induction matrix, the liver microenvironment is simulated, the cell growth factors are slowly released, and meanwhile, the anti-oxidation characteristic of quercetin is exerted, so that the induced differentiation efficiency of the embryonic stem cells is effectively improved.

The invention discloses an induced differentiation and identification method for obtaining nerve cells in different stages from mouse embryonic stem cells, and relates to the technical field of biology. The induced differentiation and identification method comprises the following steps: inoculating mouse embryonic stem cells into a culture container, and carrying out pluripotent maintenance culture by adopting a pluripotent maintenance culture medium; after the pluripotency maintenance culture is completed, discarding the culture medium, washing, and adding a nerve induced differentiation culture medium for nerve induced differentiation culture; in the process of nerve induced differentiation culture, gene marker and protein marker detection is carried out to obtain nerve cells in different stages. According to the induced differentiation and identification method provided by the invention, efficient and controllable differentiation of the mouse embryonic stem cells to the neural pedigree can be realized through standardized sequential control and a multi-dimensional verification system. According to the invention, an efficient and reliable cell source and a technical platform are provided for the research of a neurodevelopment mechanism and the construction of a neurodegenerative disease model.

The invention provides a non-coding RNA combination for inducing totipotency of mouse embryonic stem cells and application of the non-coding RNA combination, and relates to the technical field of biology. The invention provides a non-coding RNA combination for inducing totipotency of mouse embryonic stem cells. The non-coding RNA combination comprises an enhancer RNA with a sequence as shown in SEQ ID NO: 1, an enhancer RNA with a sequence as shown in SEQ ID NO: 2 and a long-chain non-coding RNA with a sequence as shown in SEQ ID NO: 3. The invention reveals that the CRISPR activation system is utilized to synergistically up-regulate the expression of the specific endogenous non-coding RNA combination for the first time, so that the mouse embryonic stem cells can be efficiently promoted to be converted into bicellular cells. Exogenous transcription factor integration is not needed, so that the safety risk of a genome is avoided; precise targeting is realized through sgRNA, and the defect of non-specific off-target of chemical small molecules is overcome; a remarkable synergistic effect is generated, and the induction efficiency of the two-cell-like cells can be remarkably improved to about 11.6%. The invention provides a safer, more efficient and controllable new strategy for stem cell fate reprogramming and early development research.

The invention relates to the technical field of biological medicines, in particular to a construction method of Gjb2 homozygous mutation deafness mice. Injecting the Gjb2 homozygous mutant mouse embryonic stem cells into a diploid mouse embryo through zona pellucida to prepare a reconstructed embryo; and transferring the reconstructed embryo to a pseudo-pregnant female mouse, so that the postpartum generation mouse is the Gjb2 homozygous mutation deafness mouse. By adopting the construction method disclosed by the invention, the survival ratio of the mouse with homozygous mutation deafness is high, a large number of important mouse models can be provided for follow-up disease treatment exploration, and an important foundation is laid for efficient establishment of mouse models with other different mutations of GJB2.

This invention discloses a method for inducing mouse embryonic stem cells to transition to a pluripotent state using H2BC21 and its application, belonging to the fields of molecular biology and stem cell engineering technology. By constructing an interference vector targeting the H2BC21 gene, packaging it with lentivirus, and infecting mouse embryonic stem cells, the expression of the H2BC21 gene is knocked down, and embryonic stem cell lines with stable low expression of H2BC21 are obtained through screening. This invention is the first to discover that H2BC21 is a core negative regulator of mammalian embryonic stem cell pluripotency. After knocking down H2BC21, the expression of pluripotency core genes Nanog, Oct4, and Sox2 is downregulated, while the expression of pluripotency core genes Zscan4b, Usp17la, and Tmem92 is upregulated. The cells exhibit molecular characteristics similar to 2-cell phase cells, successfully achieving the transition from pluripotency to a pluripotent state.

N6-methyladenosine (m6A) is the most abundant internal modification on mammalian messenger RNA (mRNA). It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein FTO. Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Aspects disclosed show that FTO mediates m6A demethylation of long-interspersed element-1 LINE1 RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m6A demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. FTO overexpression is also disclosed to modulate fertility.

The invention discloses a construction method and application of a DFNB1 type deafness mouse model with GJB2 gene frameshift mutation. The construction method comprises the following steps: (1) designing two sgRNA plasmids on a No.2 exon of a mouse Gjb2 gene; (2) screening out mouse embryonic stem cells of which the Gjb2 gene carries a c.299300delAT mutation site; (3) forming a recombinant embryo, transplanting the recombinant embryo into the uterus of the pregnant female mouse, and waiting for pregnancy and farrowing to obtain an F0-generation mouse; and (4) screening the F0 generation mice in the step (3) to obtain the mouse model carrying the c.299300delAT homozygous point mutation of the GJB2 gene. The method has the advantages that the problem of mouse embryo metaphase energy supply disorder lethal caused by Gjb2 homozygous mutation is solved, the embryo survival rate is increased, and the bottleneck of embryo lethal in model construction of a traditional gene editing technology is broken through.

The application provides an enhancer RNA molecule MZGAe1 and application thereof, and relates to the technical field of biology.The application provides an enhancer RNA molecule MZGAe1, and the nucleotide sequence of the enhancer RNA molecule MZGAe1 is shown in SEQ ID NO:1; meanwhile, the application also provides a specific sgRNA sequence for targeting and activating the molecule and a recombinant carrier thereof; by using the CRISPR activation technology, the expression of the endogenous MZGAe1 in cells can be specifically activated, and the mouse embryonic stem cells can be efficiently promoted to transform into two-cell-like cells. In the application, under the condition of endogenous activation of a single enhancer RNA molecule MZGAe1, the wild-type mouse embryonic stem cells can be efficiently transformed into two-cell-like cells, and the application provides a new efficient and specific research tool and scheme for studying zygote genome activation, early embryonic development events and cell reprogramming.

The invention discloses a construction method and application of an angel syndrome mouse model. The construction method comprises the following steps: preparing c.1435Agt carrying a maternal Ube3a gene on the basis of combination of a pilot editing technology and a tetraploid compensation technology; the invention relates to a non-human animal model of T nonsense mutation. The method comprises the following steps: co-delivering pegRNA (Ribonucleic Acid) and PEmax of a No.4 exon of a targeted Ube3a gene to a mouse embryonic stem cell, and introducing c.1435Agt at a target site; carrying out T nonsense mutation, and after tetraploid compensation technology treatment, transplanting the edited recombinant embryo to a pregnant female mouse to obtain an F0-generation mutant mouse; the accuracy of a mutation site is verified through combination of PCR amplification and gene sequencing, and a mutant strain with genetic stability is established through two generations of breeding and screening by using maternal genetic characteristics of the UBE3A gene. The invention further comprises a genotype identification method of the model animal, Ube3a gene mRNA expression quantity evaluation, behavioral evaluation and the like, and the Ube3a: c.1435 Agt is verified; the T mutant mouse model accords with the pathological characteristics of the angel syndrome, and can stably simulate the typical clinical manifestation and pathological characteristics of the angel syndrome.