CN111534481A - Method for improving efficiency of in vitro induced reprogramming of chicken embryo fibroblasts into iPS cells - Google Patents

Method for improving efficiency of in vitro induced reprogramming of chicken embryo fibroblasts into iPS cells Download PDF

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Publication number
CN111534481A
CN111534481A CN202010416986.6A CN202010416986A CN111534481A CN 111534481 A CN111534481 A CN 111534481A CN 202010416986 A CN202010416986 A CN 202010416986A CN 111534481 A CN111534481 A CN 111534481A
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glycolysis
chicken
reprogramming
ips
induced
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李碧春
袁霞
左其生
姜景译
丁颖
石祥
张明
张亚妮
孙红艳
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Yangzhou University
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Yangzhou University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"

Abstract

A method for improving the efficiency of in vitro induction reprogramming of chicken embryo fibroblasts into chicken iPS cells belongs to the technical field of biology. Earlier studies found that chicken CEF can be induced and reprogrammed to iPS by the OSNL system, but the efficiency is low, and on the basis, the role played by glycolytic metabolism in the process of inducing and reprogramming by somatic cells is researched and the induction system is further optimized. The method is simple to operate, practical and widely applicable. The chicken induced pluripotent stem cell has totipotency of development and can be induced and differentiated into various cell tissues under proper conditions, so that the chicken induced pluripotent stem cell can be used as a new stem cell source and opens up a new idea for researches on conservation of rare animals, production of transgenic animals and the like. The invention links the micromolecule compound with the cell metabolism, provides more theoretical basis and technical support for the research of inducing reprogramming of poultry somatic cells, and is also beneficial to providing more experimental materials for the research of poultry cell and embryo engineering.

Description

Method for improving efficiency of in vitro induced reprogramming of chicken embryo fibroblasts into iPS cells
Technical Field
The invention relates to a method for improving the efficiency of in vitro induced reprogramming of chicken embryo fibroblasts into chicken iPS cells, belonging to the technical field of biology.
Background
In 2006, Yamanaka introduces four transcription factors of OCT4, SOX2, KLF4 and c-MYC (OSKM) into mouse fibroblasts for the first time through a retrovirus vector to obtain pluripotent Stem Cells similar to Embryonic Stem Cells (ESCs), namely induced pluripotent Stem Cells (iPS), which have totipotency of development and can be induced to differentiate into various cell tissues under appropriate conditions. The generation of the induction somatic cell reprogramming technology not only brings a new stem cell source for people, but also opens up a new idea for researches on conservation of rare animals, production of transgenic animals and the like.
The induction technology of iPS in a plurality of species such as mice, human and the like is quite mature, but the research progress of poultry somatic cell induced reprogramming is still slow. Lu et al used NANOG, OCT4, SOX2, KLF4, C-MYC, and LIN28 (OSKMNL) lentivirus over-expression vectors to reprogram somatic cells of non-mammalian quails to iPS for the first time until 2012.
Previous studies by the applicant found that chicken CEF can be inductively reprogrammed to iPS by OCT4, SOX2, NANOG, LIN28 (OSNL) systems, but with low efficiency. Although the chicken iPS can be produced by a somatic cell induced reprogramming technology at present, the wide application of the chicken iPS is limited due to the reasons that an induction system is still unstable, the induction efficiency is low, the reprogramming mechanism is not clear and the like.
Disclosure of Invention
The invention aims to provide a method for improving the efficiency of in vitro induction reprogramming of chicken embryo fibroblasts into chicken iPS cells, aiming at the defects of the prior art.
The technical scheme of the invention is as follows:
earlier studies found that chicken CEF can be induced and reprogrammed to iPS by the OSNL system, but the efficiency is low, and on the basis, the role played by glycolytic metabolism in the process of inducing and reprogramming by somatic cells is researched and the induction system is further optimized.
To investigate how glycolytic metabolism changes during induced reprogramming, changes in glycolytic related gene expression were first detected by qRT-PCR, and changes in glycolytic levels were detected using glucose uptake and lactate production. Whether the exogenous transcription factor OSNL influences the expression of the glycolysis related gene is researched through a dual-luciferase system, and whether the glycolysis can influence the expression of the core pluripotent gene is researched through activation/inhibition of glycolysis in DF 1/ESCs.
The role of glycolysis in the process of inducing reprogramming of chicken somatic cells is determined through previous research, a proper glycolysis activator or inhibitor is screened on the basis of the research, the glycolysis activator or inhibitor is added into an OSNL induced reprogramming system, the cloning formation number of the iPS is observed, and the induction efficiency of the iPS is detected through flow.
The invention has the following beneficial effects:
the method is simple to operate, practical and widely applicable. The chicken induced pluripotent stem cell has totipotency of development and can be induced and differentiated into various cell tissues under proper conditions, so that the chicken induced pluripotent stem cell can be used as a new stem cell source and opens up a new idea for researches on conservation of rare animals, production of transgenic animals and the like.
In addition, chickens are one of the most important model organisms in the study of genetics in non-mammals. Therefore, the experimental method of the invention is not only feasible in studying somatic cell-induced reprogramming on poultry, but also applicable to other research fields of vertebrates. The invention links the micromolecule compound with the cell metabolism, provides more theoretical basis and technical support for the research of inducing reprogramming of poultry somatic cells, and is also beneficial to providing more experimental materials for the research of poultry cell and embryo engineering.
Detailed Description
Glycolysis change research in-vitro induction reprogramming process of chicken embryo fibroblast
To reprogram chicken CEF to iPS, the OCT4/SOX2/NANOG/LIN28 four-factor combination was selected to constitute the OSNL system to induce reprogramming of chicken CEF. In the induction process, the expression of endogenous pluripotency genes Oct4, Sox2, Nanog, Lin28 and a somatic cell marker gene Thy-1 is detected by means of qRT-PCR. And carrying out SSEA-1 immunofluorescence staining and alkaline phosphatase staining identification on the induced iPS.
In order to detect the change of glycolysis level in the process of inducing reprogramming by chicken somatic cells, the mRNA expression of glycolysis related genes Hk1, Pkm2, Pfkp, Ldha and Glut1 is detected by qRT-PCR, and simultaneously the glucose uptake and the lactic acid production amount in the process of reprogramming are detected.
Glycolysis function research in-vitro induction reprogramming process of chicken embryo fibroblast
In order to research whether the exogenous transcription factors can regulate glycolysis, bioinformatics prediction is utilized to discover that transcription factors OCT4, SOX2 and NANOG have common binding sites in promoter regions of glycolysis key genes Hk1, Pfkp and Ldha, and the regulation effect of the transcription factors OCT4, SOX2 and NANOG on the transcription activity of the glycolysis key genes Hk1, Pfkp and Ldha and the like is further detected through a dual-luciferase experiment.
To investigate whether glycolysis could regulate reprogramming by affecting the expression of core pluripotency factors, glycolysis activators DASA-58/inhibitors VK3 were first added to DF1/ESCs cells, respectively, and transient expression changes of pluripotency genes were detected at 0h,1h,2h, and 3h after treatment. In order to further study the effect of glycolysis on induction of chicken iPS, an inhibitor VK3 was added into a chicken embryo fibroblast in-vitro induction reprogramming system, glucose uptake and lactic acid production were detected, and induction efficiency of iPS was detected by flow.
In-vitro induction reprogramming system for optimizing chicken embryo fibroblast by adding glycolysis activator 2i
Early studies show that glycolysis plays a positive regulation role in the process of inducing reprogramming of chicken somatic cells, and a suitable small molecular compound, namely glycolysis activator 2i (PD 0325901, MEK/ERK inhibitor and SB431542, TGF-beta inhibitor) is screened on the basis of the research.
To investigate whether 2i could activate glycolysis, 2i was added to DF1 cells and cultured, and cells were harvested at 0, 12, 24, and 48h after the treatment, and qRT-PCR was performed to detect the expression of glycolysis-related genes such as Hk1, Ldha, Pkm2, and the like. In order to research whether 2i can play a role in the process of inducing reprogramming of chicken somatic cells, 2i is added into an OSNL system to induce CEF, qRT-PCR is used for detecting expression change of glycolysis related genes, glucose uptake and lactic acid production are detected, the number of chicken iPS clone formation is observed, and the induction efficiency of the chicken iPS cells is detected by flow.

Claims (3)

1. A method for improving efficiency of in vitro induction reprogramming of chicken embryo fibroblasts into iPS cells is characterized in that chicken CEF can be induced and reprogrammed into iPS through an OSNL system, a proper glycolysis activator is screened and added into the OSNL induction reprogramming system, iPS clone formation number is observed, and induction efficiency of the iPS is detected through flow.
2. The method for improving the efficiency of in vitro induced reprogramming of chicken embryo fibroblasts into iPS cells according to claim 1, wherein the effect of glycolysis on the process of in vitro induced reprogramming of chicken embryo fibroblasts is studied before screening for suitable glycolysis activators or inhibitors:
firstly, detecting glycolysis related gene expression change through qRT-PCR, and detecting glycolysis level change by utilizing glucose uptake and lactic acid production; whether the exogenous transcription factor OSNL influences the expression of the glycolysis related gene is researched through a dual-luciferase system, and whether the glycolysis can influence the expression of the core pluripotent gene is researched through activation/inhibition of glycolysis in DF 1/ESCs.
3. The method of claim 1, wherein the glycolytic activator is selected to be 2i, i.e., PD0325901 and SB 431542.
CN202010416986.6A 2020-05-18 2020-05-18 Method for improving efficiency of in vitro induced reprogramming of chicken embryo fibroblasts into iPS cells Pending CN111534481A (en)

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CN117467599A (en) * 2023-12-27 2024-01-30 广东省农业科学院动物科学研究所 Chemical inducer for reprogramming gonadal somatic cells of chickens into pluripotent stem cells of chickens and reprogramming method
CN117487743A (en) * 2023-12-27 2024-02-02 广东省农业科学院动物科学研究所 Chemical inducer for inducing chick embryo fibroblast to be chick pluripotent stem cell and induction method
CN117487743B (en) * 2023-12-27 2024-03-29 广东省农业科学院动物科学研究所 Chemical inducer for inducing chick embryo fibroblast to be chick pluripotent stem cell and induction method
CN117467599B (en) * 2023-12-27 2024-03-29 广东省农业科学院动物科学研究所 Chemical inducer for reprogramming gonadal somatic cells of chickens into pluripotent stem cells of chickens and reprogramming method

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