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Lin28 low expression mouse embryonic stem cell for maintaining Naive state of mouse embryonic stem cells

A technology of embryonic stem cells and mouse embryos, applied in the fields of biology and new medicine, can solve the problems of unclear effects of embryonic stem cells in Naive state and Primed state

Inactive Publication Date: 2017-06-16
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The effect of Lin28 on the Naive state and Primed state of embryonic stem cells cultured in vitro is still unclear

Method used

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  • Lin28 low expression mouse embryonic stem cell for maintaining Naive state of mouse embryonic stem cells
  • Lin28 low expression mouse embryonic stem cell for maintaining Naive state of mouse embryonic stem cells
  • Lin28 low expression mouse embryonic stem cell for maintaining Naive state of mouse embryonic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1, mouse RNAi-Lin28 interference vector construction

[0022] The mouse RNAi-Lin28 Ready pSIREN-RetroQ interference vector and its construction steps are as follows:

[0023] According to the BD™ Knockout RNAi Systems User Manual (No. PT3739-1), design the Lin28 RNAi interference sequence: 5'GGAGACAGGTGCTACAACT 3', and add restriction endonuclease sites EcoRI and BamHI (upstream Primer: 5'-GAT CCG GGA GAC AGG TGC TAC AAC TTT CAA GAG AAG T TGT AGC ACC TGT CTC CCT TTT TTA AGC TTG-3', downstream primer: 5'-AAT TCA AGC TTAAAA AAG GGA GAC AGG TGC TAC AAC TTC TCT TGA AAG TTG TAG CAC CTG TCT CCC G-3')

[0024] Through a series of processes such as annealing of the interference sequence primer of mouse Lin28, double digestion with the interference empty vector, connection and transformation between the interference empty vector backbone and the annealing primer; extraction of the recombinant interference plasmid, single enzyme digestion identification of the recombinan...

Embodiment 2

[0025] Example 2: Functional detection of mouse Lin28

[0026] Such as figure 2 As shown, the RNAi-Lin28 Ready pSIREN-RetroQ interference carrier was transfected into cells by liposome transfection method (see the Invitrogen instruction manual for details), and the eukaryotic resistance gene on the interference carrier was used to pass the cell culture process Add the drug puromycin to screen the mouse embryonic stem cells that have successfully recombined the vector into the genome, and expand the culture to obtain a mouse embryonic stem cell line that stably reduces the expression of Lin28, and finally pass the real-time PCR / Western blot detection method It was proved that the expression of Lin28 in the mouse embryonic stem cells transformed with the interference vector was significantly reduced at the RNA and protein levels, indicating that the cells we obtained were cell lines with significantly reduced expression of Lin28. The mouse embryonic stem cells with reduced Lin...

Embodiment 3

[0027] Example 3: Functional effect of mouse RNAi-Lin28 transgenic cells on maintaining Naive state of mouse embryonic stem cells.

[0028] Such as Figure 4 As shown, the morphology of mouse embryonic stem cells maintained in Naive state by using 2i+Lif culture conditions is similar to that of mouse embryonic stem cells with reduced Lin28 expression under normal culture conditions, and the comparison of cell morphology under the two conditions shows that Mouse embryonic stem cells with reduced Lin28 expression can maintain the Naive state of mouse embryonic stem cells.

[0029] By screening some genes specific to the Naive state of mouse embryonic stem cells that have been reported in the literature: Rex1 / Esrrb / Nr0b1 / Bmp7 / Nanog / Tfcp2l1 / Prdm14, these genes are in the Naive state of mouse embryonic stem cells at the mRNA level High expression state; use real-time PCR to detect the expression trend of these genes at the mRNA level in the mouse embryonic stem cells that stably r...

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Abstract

The invention relates to a preparation method of a Lin28 (lineage protein 28) low expression mouse embryonic stem cell for maintaining a Naive state of mouse embryonic stem cells. The method includes: constructing a Lin28 interference sequence of sequence 1 in a sequence table into an RNAi-ReadypSIREN-RetroQ interference vector so as to obtain RNA-Lin28-containing interference recombinant plasmid; and transfecting the RNA-Lin28 interference recombinant plasmid into a mouse embryonic stem cell D3 through liposome (Lipofactamine2000) transfection, and adding a drug to perform screening through an anti-drug screening gene puromycin contained in the recombinant vector and establishing a transgenic cell line, using the transgenic cell line to monitor various indicators of the mouse embryonic stem cell Naive state, thereby finding that decreased expression of Lin28 plays a promoting effect on maintenance of the mouse embryonic stem cell Naive state. The method provided by the invention can stably interfere Lin28 expression in transgenic mouse embryonic stem cells, and can obviously improve the ability of maintaining the mouse embryonic stem cell Naive state.

Description

technical field [0001] The invention relates to a method for preparing a mouse embryonic stem cell with low expression of Lin28 capable of maintaining the Naive state of the mouse embryonic stem cell, and belongs to the technical field of biology and new medicine. Background technique [0002] Lin28 (Lineage Protein 28) is an RNA and DNA-binding protein with a conserved domain that plays an important role in the maintenance of embryonic stem cell pluripotency, tumorigenesis, metabolism, and development. The effect of Lin28 as a pluripotency gene on the development of mouse embryonic stem cells can provide new ideas for the future culture methods of human and mouse embryos. [0003] Lin28 was originally discovered in Caenorhabditis elegans, through a series of gene networks to regulate the developmental period, Lin28 mutation will lead to early development of larvae, subsequent studies have found that Lin28 exists in both invertebrates and vertebrates and in Both structure...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC12N5/0606C12N2510/00
Inventor 刘娜桑慧李宗金
Owner NANKAI UNIV
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