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genetic modification method

A technology for genetic modification and zebrafish, applied in the field of genetic modification

Active Publication Date: 2021-07-27
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is currently no mature and effective microinjection technique for oocyte mothers in situ in model animals

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0031] There are strict requirements on the quality of zebrafish females and their immature eggs used for microinjection. Before the injection, the quality of the female fish and its eggs needs to be regulated to a certain extent. The specific methods are as follows: (1) Select a sexually mature female fish in a better state (zebrafish generally takes 3 months to reach sexual maturity), such as age Fish between 6 months and one year; (2) After sexual maturity, the female fish is regularly mated once a week, so that the oocytes of the female fish mature and form a relatively regular cycle; (3) In the regular After mating, let the female fish rest for two weeks to allow the oocytes to fully accumulate. Ultimately, the female fish and its oocytes are in a state that facilitates the microinjection procedure and subsequent recovery and development (see attached figure 1 ).

[0032] On the day of the injection, the morning and noon feedings are normal, but do not feed the female f...

Embodiment 2

[0036] First, using the genetic modification method described in Example 1, more than 95% of the genome of the embryo obtained by injecting the CRISPR / Cas9 system of the ntla gene has been successfully edited; while using the results obtained by existing technologies, only about 60% of the genome has been edited . And the embryos obtained by the genetic modification method described in Example 1 all have a typical tailless phenotype, while only some of the embryos obtained by the original microinjection technique have similar phenotypes, and the rest of the embryos only have shortened or almost tailless embryos. normal phenotype.

[0037] Secondly, the experimental results of the inventors using the genetic modification method described in Example 1 to simultaneously knock out 20 genome sites also showed much higher efficiency than the prior art.

[0038] Again, the inventor used the Tol2 transgenic system to construct transgenic fish on the technical platform of egg injectio...

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Abstract

The invention proposes a genetic modification method. The method comprises: in situ microinjection of genetic modification reagents to immature oocytes in model animals. Using the genetic modification method according to the embodiment of the present invention, the time of microinjection is advanced to the period when the eggs are not yet mature, and the method of direct injection in the mother is adopted. After the injected oocytes mature naturally in the mother, they are injected After excretion and fertilization, mutants with edited genomes in almost all cells can be obtained in the F0 generation, and the phenotypes consistent with the homozygous mutants can be seen in the injected contemporary embryos, as well as a stable expression of the transgene map. With technology, the time to obtain mutants is shortened to 2-3 months, and large-scale screening work is not required.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular, the present invention relates to a gene modification method. Background technique [0002] Taking zebrafish (Danio rerio) as an example, zebrafish is one of the typical model organisms in developmental biology, and its eggs undergo fertilization and early embryonic development in vitro. The female fish releases unfertilized mature eggs, while the male fish releases sperm, and the two complete the fertilization process outside the body. And the early embryos of zebrafish are transparent, which is convenient for observation and experimental operation. The existing embryo microinjection technology is microinjection to fertilized eggs successfully fertilized in vitro. It is to place the embryos that have not undergone mitosis after fertilization in a perforated injection tray made of solidified agar, and use a glass capillary pipette to heat and draw a glass needle to inject unde...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/89A01K67/027
Inventor 孟安明吴小童沈炜敏
Owner TSINGHUA UNIV
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