Method for breeding transgenic animal with improved pig growth hormone expression level
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A technology for transgenic animals and growth hormone, applied in growth hormone, biochemical equipment and methods, hormone peptides, etc., can solve problems such as embryo death, the inability to guarantee the specificity of the developmental stage of transgene expression, and the inability to conduct research.
Active Publication Date: 2010-11-24
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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Problems solved by technology
Although transgenic animals have attractive application prospects, the transgene-positive animals obtained in the early stage have sustained expression of the transgene in the early embryo, which cannot guarantee the tissue and / or developmental stage specificity of the transgene expression. When developmentally toxic or lethal, embryo death occurs early in development, making follow-up studies impossible
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Embodiment 1
[0025] Embodiment 1, the acquisition of transgenic embryo
[0026] 1. Acquisition of recombinant expression vectors
[0027] 1. Obtaining linearized TRE-GH and TET-ON
[0028] Synthesize a DNA fragment containing TRE promoter, multiple cloning sites and SV40 polyA (named TRE-MSC-SV40 polyA gene, its nucleotide sequence is shown in sequence 1 in the sequence table, synthesized by Beijing Aoke Biotechnology Co., Ltd.) . The DNA fragments synthesized above were immediately placed in ice water at 95°C for 10 minutes, and after treatment, they were double-enzymatically digested with ZraI. (purchased from Promega, Cat. No. FD3219) after ZraI. and PciI double digestion and ligation with the aforementioned TRE-MSC-SV40 polyA fragment (ligase was purchased from Promega, Cat. Quanshijin Company, Cat. No. CD201) Competent Escherichia coli; the bacterial solution was coated on an agarose gel plate, and cultured overnight in a 37°C incubator; a single colony was inoculated in liquid LB ...
Embodiment 2
[0042] Embodiment 2, the acquisition of transgenic animals and its detection
[0043] 1. Acquisition of transgenic animals
[0044] 1. Transgenic TRE-GH and TET-ON pigs
[0045] Repeat Step 1 in Step 2 of Example 1, inject the linearized TRE-GH and TET-ON into prokaryotic pig embryos, and culture them to the 8-cell stage. In vitro transvaginal cervical transplantation into the uterine horns of estrus-synchronized sows.
[0046] Genomic DNA was extracted from the ear tissue of transgenic pigs according to the kit instructions (purchased from Biotec, product number: DP1901), and pigs with integrated TRE-GH and TET-ON were screened by PCR. The PCR reaction system is 50 μl, 5 μL 10×Buffer, 8 μL 2.5 mM dNTP, 1 μL 20 μM primer PL, 1 μL 20 μM primer PR, 0.5 μL 5U / μL high-fidelity Tag polymerase, add ultrapure water to 50 μL to obtain pig genomic DNA As the template to be detected, PCR amplification program: 95°C for 5min; 94°C for 20s, annealing temperature at 56°C, 72°C for 1m, c...
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Abstract
The invention discloses a method for breeding a transgenic animal with an improved pig growth hormone expression level. The invention also discloses a method for breeding a transgenic embryo. In the method, a transgenic embryo having a higher pig growth hormone expression level than a target embryo is obtained by transplanting into a target embryo recombinant expression carriers 1) or a recombinant expression carrier 2): TRE-GH and TER-ON, and TRE-GH-TET-ON, wherein the TRE-GH is a recombinant expression carrier which is formed by inserting a DNA fragment represented by a sequence 2 in a sequence table into a multiple cloning site of a TRE carrier, and the TRE carrier is a recombinant carrier formed by inserting a DNA fragment represented by a sequence 1 in the sequence table into the multiple cloning site of a pUC plasmid; and the TRE-GH-TET-ON is a recombinant expression carrier formed by connecting a fragment of 2.5Kb, which is obtained by the double digestion of the TET-ON by Nhel and HindIII, and a fragment of 3.3KB, which is obtained by the double digestion of the TET-GH by the Nhel and HindIII. The embryo is transplanted into an animal to breed the transgenic animal. Experiments show that the GH in the blood of a transgenic pig increases sharply after the addition of DOX.
Description
technical field [0001] The invention relates to a method for cultivating transgenic animals with enhanced porcine growth hormone expression. Background technique [0002] In the 1970s, Jaenich et al. introduced SV40 DNA into mouse blastocysts, and detected SV40 DNA in offspring mouse tissues, proving that it is possible to introduce exogenous genes into embryonic cells and achieve integration. In the 1980s , Gordon et al established microinjection transgenic animal technology. Since then, the technology has developed rapidly, and various methods of establishing transgenic animals have appeared successively. Its application has penetrated into many research fields, and it has become the first choice for people to deeply understand the genetic material of organisms, study gene functions, and establish animal models of human diseases to study diseases. A powerful tool for diagnosis and treatment. Although transgenic animals have attractive application prospects, the transgene...
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