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Method for inhibiting PUMA function and improving iPS (induced Pluripotent Stem) cell construction effect

A cellular and functional technology, applied in the field of stem cells, which can solve problems such as the role of genome stability and the unclear molecular mechanism.

Active Publication Date: 2014-02-12
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PUMA is an important molecule in the downstream apoptosis pathway of p53. Inhibition or knockout of PUMA has obvious anti-radiation and anti-tumor effects in adult stem cells, but its effects on iPS generation and genome stability and its molecular mechanism are not very clear. clear

Method used

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  • Method for inhibiting PUMA function and improving iPS (induced Pluripotent Stem) cell construction effect
  • Method for inhibiting PUMA function and improving iPS (induced Pluripotent Stem) cell construction effect
  • Method for inhibiting PUMA function and improving iPS (induced Pluripotent Stem) cell construction effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 PUMA - / - Preparation, passage and cryopreservation of MEFs from mouse embryonic fibroblasts

[0041] (1) Take 8-10 weeks of PUMA - / - Mice were caged together in the evening at a ratio of 2:1 male to female, and the female mice found to have vaginal plugs were reared in single cages the next morning, and 0.5 days of embryonic development at noon was recorded as E0.5 or 0.5dpc; mouse embryos were autoclaved the day before Bacteria tweezers and scissors.

[0042] (2) Execute the pregnant mice with a pregnancy period of 13.5 days (E13.5), soak the mice in 75% alcohol, put them in a sterile ultra-clean table, cut the peritoneal cavity of the sacrificed pregnant mice with large scissors , Take out the uterus on both sides, and put the uterus in a sterile 100mm plastic petri dish with a lid.

[0043] (3) Put the removed uterus into sterile PBS and count the number of embryos.

[0044] (4) Remove the mouse embryos from the uterus step by step: remove the uterus fi...

Embodiment 2DH5

[0056] Example 2 DH5α Escherichia coli strain transformed with plasmids PMX-Oct4, PMX-Sox2, PMX-c-Myc, PMX-KLF4, and carried out small plasmid extraction and large plasmid extraction

[0057] LB medium: yeast extract 5g, tryptone 10g, NaCl 10g, dissolved in 900ml H 2 In O, adjust the pH to 7.2-7.4, add water to make up to 1L, and store under high temperature and high pressure sterilization.

[0058] LB solid medium: 5g of yeast extract, 10g of tryptone, 10g of NaCl, 15g of agar, add water to 1L, sterilize under high temperature and high pressure, lower the temperature to about 50°C, add antibiotics, pour into 100mm dish, 20ml / dish, cool and solidify Store at 4°C.

[0059] Antibiotics: Ampicillin was formulated into 100mg / mL (1000×) ampicillin (Amp) solution and stored at -20°C.

[0060] DH5α E. coli strain was purchased from Takara.

[0061] Plasmids: PMX-Oct4, PMX-Sox2, PMX-c-Myc, PMX-KLF4 were purchased from addgene.

[0062] 2.1 Bacterial Transformation Steps

[0063] (1...

Embodiment 3

[0097] Packaging, titer determination and freezing storage of embodiment 3 retroviruses

[0098] 3.1 Production of retroviruses

[0099] (1) Resuscitate Plat-E cells in a water bath at 40°C. After resuscitation, place the cells at 37°C, 5% CO 2 Cultured in an incubator, when the confluence reached 70-80%, subcultured at 1:3.

[0100] (2) Digest and count the Plat-E cells in the logarithmic growth phase. (When the confluence of Plat-E cells in a T75 flask reaches 80-90%, they can be transferred to two 100mm dishes).

[0101] (3) 8x10 per 100mm dish 6 -1x10 7 Seed the cells.

[0102] (4) Around 17-20 hours, observe whether the cell confluence reaches 90%.

[0103] (5) Replace the cell culture medium in the culture dish with 5ml fresh DMEM+10%FBS.

[0104] (6) The ratio of plasmid and lipofectamine2000 mixture (per 100mm dish) is as follows:

[0105] a) 10μl lipofectamine2000+500μl OPTI-MEM, place at room temperature for 5min;

[0106] b) 10 μg of the target plasmid (PMX...

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Abstract

The invention provides a method for inhibiting PUMA function and improving iPS (induced Pluripotent Stem) cell construction effect. The method comprises the following steps of: (1) preparing fibroblast MEF (Mouse Embryonic Fibroblast) of a PUMA- / - (PUMA gene knocked out) mice or PUMA- / + mice, and performing passage and frozen storage; (2) converting plasmid PMX-Oct 4, PMX-Sox2, PMX-c-Myc and PMX-KLF4 through DH5 alpha escherichia coli strain, and respectively highly and slightly extracting plasma; (3) packing retrovirus, tittering and performing frozen storage; (4) knocking out reprogramming of embryo fibroblasts MEF of the mice or PUMA- / + mice through PUMA gene; and (5) knocking down the programming of human fibroblast of the PUMPA function. The method has the beneficial effects that the PUMA function is inhibited and the iPS cell construction effect is improved during programming somatic cell; the stability of iPS cell genome can be protected at the same time.

Description

technical field [0001] The invention belongs to the field of stem cells, and in particular relates to a method for inhibiting the function of PUMA and improving the establishment effect of iPS cells. Background technique [0002] Simultaneously introducing four transcription factors of OCT4, SOX2, KLF4 and c-Myc into mouse skin fibroblasts can be reprogrammed to form a new type of cells similar to embryonic stem cells, called iPS cells. In 2007, iPSCs technology was successfully applied to human somatic cells. iPS cells have the multi-directional differentiation ability of embryonic stem cells, and are the best materials for research on the pathogenesis of various diseases and drug screening; they are also new hopes for the future treatment of some degenerative diseases . However, a large number of studies have shown that iPS cells induce a considerable amount of DNA damage and changes in chromosomal structure during reprogramming, which leads to the potential oncogenicity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
Inventor 程涛李彦欣
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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