Method for improving development efficiency of porcine cloned embryo

A high-efficiency technology for cloning embryos, applied to embryonic cells, biochemical equipment and methods, microorganisms, etc., can solve the problem of low development efficiency of cloned embryos

Active Publication Date: 2018-05-22
WENS FOOD GRP CO LTD +1
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  • Method for improving development efficiency of porcine cloned embryo
  • Method for improving development efficiency of porcine cloned embryo
  • Method for improving development efficiency of porcine cloned embryo

Examples

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Embodiment 1

[0028] Embodiment 1 The method for improving the developmental efficiency of pig cloned embryos of the present invention

[0029] 1: Collection method of sow uterine fluid

[0030] Select a 24-month-old healthy sow identified as 5 days after estrus in the pig farm, remove the uterus after slaughter, and transport it to the laboratory within 2 hours. First wash the surface with normal saline, then use scissors to cut the uterus from the umbrella of the fallopian tube to the uterine horn, and use the rubber tube of a small vacuum pump in the molecular laboratory to connect the tip of a 200μl pipette along the inner wall of the sow’s uterus to absorb the surface of the endometrium. liquid and collected into 15ml centrifuge tubes. By this method, about 10-15ml of uterine fluid can be collected from the uterus of a sow. Thereafter, filter-sterilize with a 0.2 μm diameter filter. Freeze at -80°C, valid for more than one year. Calculated by adding sow uterine fluid with a volume ...

Embodiment 2

[0042] Example 2 Detection of Gene Expression and Cloning Efficiency of Pig Cloned Embryos Cultured with Sow Uterine Fluid

[0043] 1: The specific steps of the collection method of sow uterine fluid, the in vitro maturation culture of pig oocytes, the culture of pig skin fibroblasts and the method of somatic cell nuclear transfer are the same as in Example 1.

[0044] 2: Comparative analysis of gene expression in early embryos

[0045] 70 cloned embryos in the control group and cloned embryos treated with uterine fluid at a volume concentration of 1% were constructed, cultured for 48 hours, and 5 cloned embryos divided to the 4-cell stage were selected for transcriptional library and sequencing of individual embryos. At the same time, a sow of the same strain was bred with the semen of the same boar from which the cloned embryos were constructed, and the sow's uterus was slaughtered about 48 hours after mating and transported to the laboratory. Fix the front end of the uteri...

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Abstract

The invention relates to the field of animal cloning methods, in particular to a method for improving the development efficiency of a porcine cloned embryo. The method mainly comprises the step of adding a uterine fluid of a sow into a basic culture solution for the porcine cloned embryo. By the method, the blastocyst rate during early development of the cloned embryo and the total number of cellsin a blastocyst are significantly increased; a transcriptome sequencing result of the early embryo shows that culture through addition of the uterine fluid of the sow reduces the number of differentially expressed genes between the cloned embryo and a normal in-vivo fertilized embryo; most importantly, through the addition of the uterine fluid of the sow, also increased the fertility rate of therecipient sow of the cloned embryos and the number of litters per litter, the parturition rate of a receptor sow with the cloned embryo and the number of live births are also increased, which means that by the method, the final efficiency of the in-vivo development of the cloned embryo is effectively improved.

Description

technical field [0001] The invention relates to the field of animal cloning methods, in particular to a method for improving the development efficiency of pig cloned embryos. Background technique [0002] Mammalian somatic cell nuclear transfer technology is a new technology with broad application prospects in the fields of agriculture and regenerative medicine. For example, in 2000, Polejaeva et al. reported for the first time the use of adult somatic cell nuclear transfer to prepare cloned pigs; in 2004, Lai&Prather published the pig body Operation procedure of nuclear transfer technology. Since the birth of the world's first somatic cell cloned mammal "Dolly" sheep, a large number of animals have been successfully cloned. It has important application value, but the whole-stage developmental efficiency of somatic cell cloned embryos (the number of cloned offspring at birth / the number of reconstructed cloned embryos) is much lower than that of naturally fertilized embryos,...

Claims

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Application Information

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IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2500/84
Inventor 贺晓燕吴珍芳谈成蔡更元赵成法石俊松周荣
Owner WENS FOOD GRP CO LTD
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