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Method and culture medium for cultivating transgenic animal embryo cells or transgenic animals

A technology for transgenic animals and embryonic cells, applied in the field of a method and culture medium for cultivating transgenic monkeys, to achieve the effect of improving in vitro development and increasing the accuracy of gene modification

Active Publication Date: 2017-12-05
广州元曦生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no studies have reported whether single blastomeres isolated from four-cell stage embryos in primates can successfully hatch to the blastocyst stage in vitro or whether single blastomeres derived from four-cell stage embryos can be transplanted. Successfully developed a baby monkey

Method used

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  • Method and culture medium for cultivating transgenic animal embryo cells or transgenic animals
  • Method and culture medium for cultivating transgenic animal embryo cells or transgenic animals
  • Method and culture medium for cultivating transgenic animal embryo cells or transgenic animals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 transgenic process

[0060] Construction of CRISPR / Cas9 sgRNA vectors containing PINK1 gene and ASPM gene target sequences. The sgRNA expression vector contains a T7 promoter to control the expression of sgRNA, and can be inserted into a specific target sequence of 20bpsgRNA after digestion with BbsI restriction enzyme, the recognition site of the specific target sequence is located in the second exon of PINK1 gene, ASPM gene third exon and tenth exon. In order to screen the sgRNA sequences of PINK1 and ASPM genes capable of gene editing and with small off-target effects, the applicant used NCBI's Blast software to screen the PINK1 gene sequences of rhesus monkeys and the ASPM gene sequences of cynomolgus monkeys with low off-target effects and conforming to CRISPR / Cas9 binds to the target sequence site of the genomic feature (5'-G(19N)-NGG3') or (5'-CCN(19N)-C-3'), as shown in Table 1.

[0061] a. Chemically synthesize oligonucleotide chains and synthes...

Embodiment 2

[0078] Example 2 Isolation of single blastomeres

[0079] 1. Eggs obtained by superovulation were subjected to single sperm injection (ICSI) and after double pronuclei appeared in the embryo, fertilization was confirmed. 8-10 hours after single sperm injection, Cas9 / gRNA was injected into the cytoplasm of the fertilized egg.

[0080] 2. Culture in conventional HECM9 medium (not the modified medium used before blastomere isolation) for 1-2 days until the embryo develops to four cells, and then digest it with 0.5% (w / v) pronase for 45-60 seconds to remove Cellophane tape.

[0081] 3. Transfer the embryos with the zona pellucida removed to 0.05% trypsin drops, digest for 2-3 minutes, separate the four-cell embryos with the zona pellucida removed, and wash twice with HECM9 to eliminate residual trypsin on the blastomeres influences.

Embodiment 3

[0082] Example 3 Culture and Differentiation and Development of Single Blastomeres

[0083] 1. Simply put the blastomeres into the pre-made empty zona pellucida, transfer them to the modified HECM9 medium and culture them for 1-2 days to develop into 4 cells again.

[0084] (1) Conventional culture medium, i.e. traditional HECM9 medium, has adopted the formula shown in Table 2 in this exemplary embodiment;

[0085] (2) Improved HECM9 medium: 2 mmol of dextrose and 5 mmol of vitamin C were added to the formulation in Table 2.

[0086] The pH of the medium is PH7.1; the osmotic pressure is 285.

[0087] Culture conditions: the gas ratio is 75% nitrogen, 5% carbon dioxide, 20% oxygen, and the culture temperature is 37 degrees Celsius

[0088] Table 2. Traditional HECM9 medium formula

[0089] Element

mg / 100ml

PVA

10.0

Phenol red

1.0

CaCl 2 2H 2 o

28.0

MgCl 2 .6H 2 o

9.3

NaCl

664.0

KCl

22.3

...

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Abstract

The invention belongs to the field of biotechnologies, and relates to a method and culture medium for culturing transgenic animal embryonic cells or transgenic animals. The method comprises the steps that animal fertilized eggs obtained after gene modification are cultured to the four-cell stage, a zona pellucida is removed, then plasmodesmata are removed, four-cell embryos are separated into four single blastomeres, then the single blastomeres are put back into the zona pellucida with cytoplasm removed, in-vitro culture is carried out, and homozygous gene modified embryos are obtained efficiently. The optimized culture medium is provided at the same time. Based on the embryo blastomeres separation and independent culture technology, blastospheres can be successfully obtained in an optimized embryo culture system, and the efficiency is 30.33%. In addition, the homozygosis targeting efficiency of the obtained regenerated embryos is relatively improved obviously relative to that of unsplit developing embryos, and the homozygous mutation efficiency reaches up to 55.80%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method and culture medium for cultivating transgenic monkeys. technical background [0002] Animal disease models play a key role in the study of human disease pathogenesis and drug screening. Gene editing methods for establishing animal models mainly include transgenic and gene targeting. Genetic modification is the introduction or integration of exogenous genes into the genome by artificial methods, and can be stably passed on to the next generation. However, relying on virus methods to genetically modify animals also has certain limitations, that is, the random insertion of the transferred gene makes the mutation site unpredictable and the number of mutations uncontrollable, which limits the establishment of animal models. Gene targeting technology refers to changing a specific gene in the genome through endogenous DNA site-specific recombination to study the function of the gen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10A01K67/027
CPCA01K67/0275C12N5/0604C12N2500/05C12N2500/30C12N2500/34C12N2500/38C12N2500/50C12N2501/999
Inventor 李晓江涂著池杨伟莉闫森刘旭东郭祥玉
Owner 广州元曦生物科技有限公司
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