New method for improving transgenic efficiency of animals
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A transgenic and new method technology, applied in the new field of improving the efficiency of animal transgenics, can solve the problems of huge differences in the positive rate of transgenic animals and low efficiency
Inactive Publication Date: 2011-09-14
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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Problems solved by technology
In the 1990s, this technology has been commercialized, and some companies can even provide transgenic animals that integrate foreign genes, but the efficiency is still too low, and only 4% of oocytes can continue to develop after injection
In recent years, the use of sperm as a carrier to produce transgenic animals has become a research hotspot, but the positive rate of transgenic animals produced by this method varies greatly, ranging from 4% to 90%, so it is imperative to verify and optimize this method
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Embodiment 1
[0036] Example 1 Obtaining transgenic offspring mice by spot injection of the mother
[0037] 1. Preparation of experimental materials and reagents
[0038] 1. Experimental materials
[0039] Kunming white mice aged 5-8 weeks were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences. A total of 180 female mice and 90 male mice were used in the experiment. Trypan blue stain, pentobarbital sodium, PMSG, HCG, human penicillin / streptomycin, tryptone, yeast extract, DEPC (diethylpyrocarbonate), maleic acid, Tween-20, SDS, Agar powder, DH5α competent cells, Lipofectin2000, 2×Taq PCR MasterMix, X-gal, IPTG, One-Step RT Kit, DIG High Prime DNA Labeling and Detection StarterKit 1, Agarose Gel Recovery Kit, Tissue Genomic DNA Extraction Kit; enhanced fluorescent protein particles: pIRES-eGFP, endotoxin-free plasmid extraction kit, endonuclease BamH I, endonuclease Xho I, M2 medium.
[0040] 2. Reagent preparation
[0041] 1) Physiological saline:...
Embodiment 2
[0185] Example 2 Obtaining transgenic offspring sheep by dot injection on the mother generation
[0186] According to the above experimental procedure, the present invention successfully transfers the inhibin gene into the testis tissue and ovary tissue of sheep respectively through spot injection. The offspring obtained by mating male sheep with testes and normal female sheep were called group A, and the offspring obtained by mating female sheep with ovary injection and normal male sheep were called group B, the testes of male sheep and the ovaries of female sheep were equal After the operation, the offspring obtained from mating are called group C. The comparison results of the PCR positive rate of the offspring of the sheep in the three experimental groups are as follows: The positive rate of the tail tip tissue of the F1 generation sheep in the A group was 46.2% (6 / 13). The positive rate of PCR detection of tail tip tissue of F1 sheep in group B was 38.5% (5 / 13), and the ...
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Abstract
The invention discloses a new method for improving transgenic efficiency of animals. In the transgenic method, the sperm cells and the oocytes are used as vectors, through improvement of the method, the exogenous genes are respectively led to the sperm cells and the oocytes and the exogenous genes carried by the sperm cells and the oocytes are further integrated in the embryos through fertilization process, thus improving the efficiency of leading the exogenous genes to the nuclei and then realizing high efficiency expression. The method is characterized by respectively transfecting female and male reproductive cell systems by adopting a point injection method before the female and male mice mate with each other and then selecting the male mice 1-5 days and 35-40 days after surgery and the female mice 1-7 days after surgery to mate with each other to obtain the transgenic mouse offspring. By adopting the method, transgenosis of sheep is explored, the transgenic efficiency of big mammals can be improved and a new approach is provided for breeding in the animal husbandry. In the method, a transgenic system adopting female and male reproductive cells for joint mediation is explored.
Description
technical field [0001] The present invention relates to methods of transgenesis. Specifically, the present invention relates to a transgenic method using sperm and oocytes as carriers, introducing exogenous genes into sperm and oocytes respectively, and further integrating the exogenous genes carried by the two into embryos through the fertilization process, so as to Improve the efficiency of exogenous genes into the nucleus. Background technique [0002] Transgenic animals use experimental methods to introduce the required target gene into the fertilized eggs of animals, so that they can proliferate and be stably passed on to offspring. At present, scientists have successfully obtained transgenic mice, rabbits, chickens, goats, pigs, sheep, cattle, frogs and various transgenic fish. Transgenic animals are a new milestone in the development of genetics, showing great advantages and broad application prospects in the study of gene function and expression, the establishment ...
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Application Information
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63A01K67/02
CPCA01K67/0275A01K2227/105C12N15/8509
Inventor 苗向阳
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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