Method for preparing feed layer cells by using R6-MEF carried with Xist Tale suppression transcription factor R6

A R6-MEF, feeder cell technology, applied in genetically modified cells, biochemical equipment and methods, artificially induced pluripotent cells, etc., can solve cytoplasmic vacuoles, affect feeder layer quality, and change cell functions problems, to achieve the effect of good clone shape, high pluripotency gene expression level, and fast growth rate

Active Publication Date: 2018-07-06
INNER MONGOLIA UNIVERSITY +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

STO cells can be cultured in vitro for a long time, easy to use, and widely used. However, if STO cells are continuously passaged in vitro for a long time, abnormal phenomena such as cytoplasmic vacuoles and dense body formation will often occur, resulting in changes in cell function and affecting the quality of the feeder layer.

Method used

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  • Method for preparing feed layer cells by using R6-MEF carried with Xist Tale suppression transcription factor R6
  • Method for preparing feed layer cells by using R6-MEF carried with Xist Tale suppression transcription factor R6
  • Method for preparing feed layer cells by using R6-MEF carried with Xist Tale suppression transcription factor R6

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Experimental program
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preparation example Construction

[0041] (2) Preparation of R6-MEF cell line:

[0042] A. Lipofection of Oct4-GFP MEFs:

[0043] Culture MEFs carrying Oct4-GFP in M10 culture medium in a 6-well plate, and transfect when Oct4-GFP MEFs reach 40%-60% confluence, replace with 2 mL of fresh M15 culture medium 30 minutes before transfection, put Equilibrate in the incubator for 30 minutes, add 500 μL transfection solution prepared and mixed in advance to the M15 culture medium, and place at 37°C, 5% CO 2 Cultivate in an incubator for liposome transfection for 12-18 hours;

[0044] B. Induction and establishment of R6-MEFs after liposome transfection:

[0045] The next day after liposome transfection, replace the M15 culture medium with M15+Dox culture medium and culture at 37°C, 5% CO 2 Culture in an incubator; the third day after liposome transfection, the transfection efficiency was detected by fluorescence microscope: the successfully transfected cells expressed red fluorescent protein, replaced with new M15+D...

Embodiment 1

[0071] Embodiment 1: Preparation of Oct4-GFP MEFs

[0072] Sexually mature C57BL / 6J female mice and MF1, 129 / sv male mice carrying Oct4-GFP were caged together at a ratio of 1(♂):2(♀), and the fetuses were obtained by caesarean section from the 13.5-day-old pregnant mice and washed. After cleaning, remove the head, limbs and tail, wash and cut into pieces, digest with 0.25% Trypsin-EDTA digestion solution (25200056, Gibco) in a 37°C water bath for 20min, and mix several times during the period; continue to add 0.25% Trypsin-EDTA EDT A digestion solution (25200056, Gibco), digested in a water bath at 37°C for 20 minutes; pipetting and mixing, adding M10 culture medium to stop digestion, centrifuging at 1000rpm for 10 minutes, discarding the supernatant, adding an appropriate amount of M10 culture medium, repeating pipetting about 10 times, Place in a T25 culture flask at 37°C, 5% CO 2 Cultivate in an incubator, and after reaching 80% confluence, carry out 1:3-1:5 subculture an...

Embodiment 2

[0073] Embodiment 2: Preparation of R6-MEF cell line

[0074] A. Lipofection of Oct4-GFP MEFs:

[0075] Culture MEFs carrying Oct4-GFP in M10 culture medium in a 6-well plate, and transfect when Oct4-GFP MEFs reach 40%-60% confluence, replace with 2 mL of fresh M15 culture medium 30 minutes before transfection, put Equilibrate in the incubator for 30 minutes, add 500 μL transfection solution prepared and mixed in advance to the M15 culture medium, and place at 37°C, 5% CO 2 Cultivate in an incubator for liposome transfection for 12-18 hours;

[0076] B. Induction and establishment of R6-MEFs after liposome transfection:

[0077] On the second day after liposome transfection, replace the M15 culture medium with M15+Dox culture medium and culture in a 37°C, 5% CO2 incubator; on the third day after liposome transfection, detect the transfection efficiency with a fluorescence microscope : Successfully transfected cells express red fluorescent protein, replace with new M15+Dox+p...

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Abstract

The invention discloses a method for preparing feed layer cells by using R6 transfected MEF. The method comprises the following steps: firstly using suppression transcription factor R6 transfected MEFbased on transcriptional activation effect material sample Tale combined inside an Xist first intron area to obtain immortal type MEF cell lines R6-MEF, after screening the various R6-MEF cell lines,selecting the R6-MEF cell lines with relatively high multipotential gene expression, and after mitomycin treatment, obtaining the novel feed layer cells R6-feeder, so that the obtained feed layer cells can be preferably used for maintaining the pluripotency iPSC of embryonic stem cells ESC and induced multipotential stem cells. According to the method, a new selection is provided for cultivationand research of stem cells, and a new technological and theoretical basis is provided for researching how to obtain and improve iPSCs of people, pigs, cows and other mammals.

Description

technical field [0001] The invention belongs to a preparation method of a novel feeder layer cell suitable for culturing pluripotent stem cells, and particularly relates to a method for preparing feeder layer cells by using R6-MEF carrying Xist Tale inhibitory transcription factor R6. Background technique [0002] Embryonic Stem Cell (ESC) refers to a kind of pluripotent cell selected from the inner cell mass of early embryo or primordial germ cell by in vitro differentiation inhibition culture. It has the characteristics of unlimited proliferation, self-renewal and multi-directional differentiation in vitro, and can be induced to differentiate into all types of adult cells in the body no matter in vivo or in vitro. Induced pluripotent stem cells (iPSC) are pluripotent stem cells that directly reprogram adult cells into embryonic stem cells (ESC) by introducing specific transcription factors. iPSC also has the totipotency of self-renewal and differentiation, and its functio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0735C12N5/074
CPCC07K14/47C12N5/0603C12N5/0606C12N5/0656C12N5/0696C12N2510/00
Inventor 张金吨李喜和梁延峰范丽红
Owner INNER MONGOLIA UNIVERSITY
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