In-vitro maturation culture method for oocyte of mouse and method for establishing parthenogenetic embryonic stem cell line

A technology for in vitro maturation and culture of oocytes, which is applied in the field of in vitro maturation and culture of mouse oocytes and the establishment of parthenogenetic embryonic stem cell lines. It can solve the problems of complex components, high formula costs, and low egg maturation, and achieve low cost. , the effect of simple ingredients

Inactive Publication Date: 2010-06-16
SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a mouse egg with a simple and effective formula and an ovum maturation rate of more than 83% to solve the problems of high formula cost, complex ingredients and low egg maturation in the prior art. Blast cell maturation culture method in vitro

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  • In-vitro maturation culture method for oocyte of mouse and method for establishing parthenogenetic embryonic stem cell line
  • In-vitro maturation culture method for oocyte of mouse and method for establishing parthenogenetic embryonic stem cell line
  • In-vitro maturation culture method for oocyte of mouse and method for establishing parthenogenetic embryonic stem cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Collection of mouse immature eggs and in vitro maturation culture

[0046] First prepare mouse immature egg culture fluid according to formula: 95 volume % MEM (purchased from Invitrogen), 5 volume % fetal calf serum (purchased from Hyclone), 0.24mM sodium pyruvate (purchased from sigma), 1.5units / mlHGG ( available from sigma), 1 unit / ml PMSG (available from sigma).

[0047] Inject pregnant horse serum gonadotropin (PMSG) into female mice according to a dose of 5IU / only, and after 42 to 46 hours, the mice were killed by breaking the spine, and the mouse ovaries were taken out and placed in a place containing 20mM HEPES (HEPES is commonly used in the art). culture medium, commercially available) immature egg culture medium, the cumulus-oocyte complex in the follicle in the ovary was taken out with a small needle, observed under a microscope to confirm that it was a GV stage egg, and placed in a preheated ( >1 hour) in immature egg culture solution, the culture...

Embodiment 2

[0049] Example 2 Isolation of Parthenogenetic Embryonic Stem Cells

[0050] The KSOM medium in this example is commercially available.

[0051] Move the mature mouse eggs cultured in vitro in the above Example 1 to the preheated (>1 hour) Ca 2+ -Activate in free KSOM medium for 4 hours, then transfer to KSOM medium for culture, observe the activation and development of eggs for about 24 hours, and observe the development of eggs to form blastocysts after 96 hours of culture, and transfer the blastocysts to mitomycin treatment There are about 10 blastocysts per well on a four-well plate of the feeder fibroblasts. The formula of the culture medium used here is: Knockout DMEM (purchased from Invitrogen) is used as the base liquid, and Knockout serum substitute (KSR, purchased from Invitrogen), 1000IU / ml leukemia inhibitory factor, 1mM L-glutamine (purchased from Invitrogen), 0.1mM MEM non-essential amino acids (purchased from Sigma), 0.1mM β-mercaptoethanol (purchased from Sigm...

Embodiment 3

[0057] Example 3 Research on Pluripotency of Mouse Parthenogenetic Embryonic Stem Cell Lines

[0058] Observing the mouse parthenogenetic embryonic stem cells obtained in Example 2 and the stem cells derived from fertilized eggs, it can be found that although the embryonic stem cells in Example 2 are matured and cultivated in vitro by immature eggs and then parthenogenetically activated and developed, they and fertilized eggs There is no difference in the shape and growth speed of the derived stem cells, and the karyotype analysis also shows 40 normal items, such as image 3 shown.

[0059] OCT-4 is a transcription factor that can be expressed in the nucleus. In this example, both the mouse parthenogenetic embryonic stem cells obtained in Example 2 and the stem cells derived from fertilized eggs were expressed with OCT-4, and the results were as follows image 3 As shown, the mouse parthenogenetic embryonic stem cells obtained in Example 2 and the stem cells derived from fer...

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Abstract

The invention discloses an in-vitro maturation culture method for an oocyte mouse and a method for establishing a parthenogenetic embryonic stem cell line. In the in-vitro maturation culture method, an immature oocyte is cultured in a basic culture solution in which the HCG (human chorionic gonadotropin) and PMSG (pregnant mare serum gonadotropin) are added, and the maturation rate of the oocyte can reach more than 83 percent. In the method for establishing a parthenogenetic embryonic stem cell line, the parthenogenetic activation culture development is carried out on the mouse oocyte after in-vitro maturation culture to obtain the embryonic stem cell line; a hepatocyte obtained by the method has no immunogenicity, is simultaneously equivalent to a stem cell derived from a fertilized embryo on totipotency and has guiding significance for utilizing in-vitro maturation of the immature oocyte of a human and separating parthenogenetic embryonic stem cells. Aiming at the current conditionsof human ovum donation shortage, great discarding of clinical immature oocytes by an assisted reproductive technology, and the like, the two methods are combined to lay the foundation for developing and utilizing the in-vitro maturation culture of the immature oocyte of people and further researching the parthenogenetic embryo by utilizing an in-vitro mature ovum and separating the embryonic stemcells.

Description

technical field [0001] The invention relates to the technical field of embryonic stem cell lines, in particular to a method for in vitro maturation and culture of mouse oocytes and a method for establishing a parthenogenetic embryonic stem cell line. Background technique [0002] Direct activation of M II eggs in the absence of the male gamete genome enables the formation of parthenogenetic blastocysts from which parthenogenetic embryonic stem cells can be isolated. Parthenogenetic embryonic stem cells have the general characteristics of embryonic stem cells derived from fertilized embryos. [0003] The currently established embryonic stem cell lines are mainly derived from blastocysts, and the isolation of the inner cell mass of blastocysts is very important. The methods for isolating blastocyst inner cell mass mainly include immunosurgery and mechanical methods. During the isolation process of embryonic inner cell mass (ICM), choosing a method that least damages the abili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075C12N5/0735C12R1/91
Inventor 刘林刘忠周玲君吕祁峰黄军就
Owner SUN YAT SEN UNIV
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