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Human parthenogenetic embryo stem cell line with two active X chromosomes and derivatives thereof

A technology of embryonic stem cells and X chromosome, applied in the field of human parthenogenetic embryonic stem cell lines and its derivatives, can solve the problems of X chromosome inactivation failure and unknown mechanism, and achieve the effect of avoiding immune rejection and broad application prospects

Inactive Publication Date: 2009-09-09
广州医学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The selection and initiation of X chromosome inactivation occurs at the blastocyst stage. This process is controlled by the X inactivation center (XIC). XIC is a cis-acting site that contains information to identify the number of X chromosomes and the XIST gene. One chromosome is active, but the mechanism is unknown, the loss of XIST gene will lead to the failure of X chromosome inactivation

Method used

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  • Human parthenogenetic embryo stem cell line with two active X chromosomes and derivatives thereof
  • Human parthenogenetic embryo stem cell line with two active X chromosomes and derivatives thereof
  • Human parthenogenetic embryo stem cell line with two active X chromosomes and derivatives thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Acquisition of human parthenogenetic embryonic stem cells

[0038] 1. Volunteer recruitment and relevant informed consent

[0039] This experiment strictly followed the relevant regulations of the Ministry of Health of the People's Republic of China (No.460, 2003).

[0040] All volunteers were informed of the experimental procedures in detail, including the use of eggs and the purpose of the research, and voluntarily signed a series of informed consent forms without any economic benefits. It is guaranteed that all eggs will be used for basic research and will not be used for reproductive purposes.

[0041] 2. Superovulation induction and egg harvesting of volunteers

[0042] Each volunteer must undergo a strict physical examination before entering the superovulation induction cycle, including AIDS, hepatitis B, hepatitis C, sexually transmitted diseases and other infectious diseases. Qualified persons adopt the long program or the short program to induce sup...

Embodiment 2

[0086] Example 2 Identification of phES cells

[0087] 1. Routine identification of phES cells

[0088] 1. Alkaline phosphatase detection (AKP staining)

[0089] When the phES cells were cultured to the 20th passage, alkaline phosphatase detection (AKP staining) was carried out: remove the culture medium, fix with 4% paraformaldehyde for 15 minutes, rinse twice with PBS, add alkaline phosphatase incubation solution, and keep at room temperature Incubate for 30 minutes until staining is suitable, rinse with PBS, and observe the results under a light microscope.

[0090] 2. Identification of human embryonic stem cell surface antigens

[0091] (1) The phES cells were subcultured to the 4th day, and the culture was terminated when the growth was vigorous, and fixed with 4% paraformaldehyde for 20 minutes.

[0092] (2) Rinse twice with PBS, 5-10min each time

[0093] (3) 0.1% Triton X-100 / PBS for 10 minutes

[0094] (4) Rinse twice with PBS, 5-10 minutes each time.

[0095] (...

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Abstract

The invention relates to a human parthenogenetic embryo stem cell line with two active X chromosomes and derivatives thereof. A karyotype of the human parthenogenetic embryo stem cell line is 46, XX before 10 generations; the karyotype of the cell line becomes a chimera from the 20th generation; all cells of the cell line lose an active X chromosome at the 35th generation; and in the inactivation detection of the X chromosome, only the state of the active X chromosome is maintained all along, but the presence of the inactivated X chromosome is not discovered. The parthenogenetic activation can be achieved by adopting an artificial activation condition causing a second polar body to be discharged; therefore, the human parthenogenetic embryo stem cell line and the derivatives thereof can be obtained. The cell line and the derivatives thereof have important significance on researching the characteristics of the cell on genetics and epigenetics and further applying the cell to clinically treating certain diseases; and a cell source for cell replacement therapy can be obtained by transforming genes of the cell or modifying the genes on the epigenetics.

Description

technical field [0001] The present invention relates to pluripotent embryonic stem cells, in particular to a human parthenogenetic embryonic stem cell line and its derivatives. Background technique [0002] As we all know, the so-called parthenogenesis is a process in which an egg cell develops into an embryo spontaneously or through physical and chemical stimulation without sperm. That is, the embryo produced by the female gamete without any action of the male gamete. Regardless of whether it develops into an individual, it is called parthenogenesis. Parthenogenesis in nature is a reproductive phenomenon, and parthenogenesis also exists in reptiles, fish, and birds. [0003] Although mammals cannot reproduce in this way, they can simulate the fertilization process through artificial stimulation in vitro, so that oocytes can be activated without sperm, thereby obtaining parthenogenetic embryos. This is an important biotechnology that can be used to indirectly assess the q...

Claims

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Application Information

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IPC IPC(8): C12N5/08
Inventor 孙筱放
Owner 广州医学院
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