Editing method aiming to female genes

A gene editing and editing technology, which is applied to other methods of inserting foreign genetic materials, genetic engineering, plant gene improvement, etc., can solve the problems of editing failure, loss of use value of cells, and inability to predict which cells, etc., to facilitate transformation, Ensure the effect of the effect

Active Publication Date: 2018-09-07
陈子江 +1
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  • Summary
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, for embryonic gene editing, we cannot determine the editing efficiency without testing all the cells of the embryo, so we cannot predict which cells are successfully edited and which cells fail to edit before testing.
But once molecularly tested, the cells are useless, which is fatal to the embryo itself
On the other hand, under the existing technical conditions, it is impossible to carry out related gene editing on mammalian female chromosomes, which further limits the scope of application of gene editing technology in the future

Method used

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  • Editing method aiming to female genes
  • Editing method aiming to female genes
  • Editing method aiming to female genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. Acquisition of parthenogenetic fertilized eggs: Mouse MII stage oocytes are parthenogenetically activated with calcium ionophore A23187 combined with puromycin to form parthenogenetic fertilization containing only female chromosomes (ie, haploid parthenogenetic chromosomes) eggs (see figure 1 1 and figure 2 Middle 9).

[0053] 2. Parthenogenetic zygote gene editing: use CRISPR / Cas9 gene editing technology to edit the target gene of the parthenogenetic zygote obtained in step 1 at the pronuclear stage (see figure 1 Medium 2).

[0054] 3. Isolation of blastomeres from edited parthenogenetic embryos: The edited parthenogenetic zygotes obtained in step 2 were further subjected to embryo culture until the 8-cell stage (see figure 1 Middle 3). Aspirate one blastomere from the embryo with a 30 µm inner diameter embryo biopsy needle (see figure 1 Middle 4).

[0055] 4. Reconstruct the parthenogenetic embryo: remove the spindle body of the mouse MII stage oocyte (includi...

Embodiment 2

[0059] 1. Acquisition of parthenogenetic fertilized eggs: Inject the mouse MII stage oocytes with mouse single sperm to activate fertilization. After the double pronuclei appear (pronuclear stage), the male pronuclei are removed and the female pronuclei are retained, and finally a single pronucleus is formed. A parthenogenetic zygote containing female chromosomes (see figure 1 1 and figure 2 Medium 10).

[0060] 2. Parthenogenetic zygote gene editing: use CRISPR / Cas9 gene editing technology to edit the target gene of the parthenogenetic zygote obtained in step 1 at the pronuclear stage (see figure 1 Medium 2).

[0061] 3. Isolation of blastomeres from edited parthenogenetic embryos: The edited parthenogenetic zygotes obtained in step 2 were further subjected to embryo culture until the 8-cell stage (see figure 1 Middle 3). Aspirate one blastomere from the embryo with a 30 µm inner diameter embryo biopsy needle (see figure 1 Middle 4).

[0062] 4. Reconstruct the parthen...

Embodiment 3

[0066] 1. Acquisition of parthenogenetic zygotes: Mouse MII stage oocytes were parthenogenetically activated using calcium ionophore A23187 combined with puromycin to form parthenogenetic zygotes containing only female chromosomes (see figure 1 1 and figure 2 Middle 9).

[0067] 2. Parthenogenetic zygote gene editing: use CRISPR / Cas9 gene editing technology to edit the target gene of the parthenogenetic zygote obtained in step 1 at the pronuclear stage (see figure 1 Medium 2).

[0068] 3. Isolation of blastomeres from edited parthenogenetic embryos: The edited parthenogenetic zygotes obtained in step 2 were further subjected to embryo culture until the 8-cell stage (see figure 1 Middle 3). Aspirate one blastomere from the embryo with a 30 µm inner diameter embryo biopsy needle (see figure 1 Middle 4).

[0069] 4. Reconstruction of parthenogenetic embryos: Inject the blastomeres drawn in step 3 into the perivitelline space of mouse MII stage oocytes, activate and fertiliz...

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Abstract

The invention discloses an editing method aiming to female genes. The editing method comprises the following steps of acquisition of parthenogenetic fertilized eggs, editing of the parthenogenetic fertilized eggs, separation of edited parthenogenetic embryo blastomeres, reconstruction of parthenogenetic embryos, and identification of an editing result. The method provided by the invention can indirectly realize a function of performing precise editing on mammal ovum, can ensure the final gene editing effect and is beneficial to conversion of a gene editing technology to clinical medicines.

Description

technical field [0001] The present invention relates to a method for editing female genes, and more specifically, relates to a method for editing female genes by combining gene editing technology with nuclear transfer technology. Background technique [0002] Gene editing technology is currently making more and more progress in the research field, and large-scale attempts have been made in human cell samples. However, at present, the main risks of gene editing technology are still editing efficiency and off-target issues, which are particularly critical for clinical promotion and use. Only when the related risks are overcome can it be safely promoted to the clinic. At present, for embryonic gene editing, we cannot determine the efficiency of editing without testing all the cells of the embryo, so we cannot predict which cells are successfully edited and which cells are unsuccessfully edited before testing. But once molecularly tested, the cells are useless, which is fatal t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/873C12N15/90
CPCC12N9/22C12N15/873C12N15/907
Inventor 陈子江吴克良赵涵马金龙
Owner 陈子江
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