Method for building immortalized preadipocytes of chicken

A preadipocyte and immortalization technology, which is applied in the field of establishing immortalized preadipocytes in chickens, can solve the problems of heterogeneity, differences in cytogenetic backgrounds from different sources, and difficulty in obtaining stable and reliable results, so as to maintain contact inhibition. Effect

Active Publication Date: 2014-06-11
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0005] The present invention aims to solve the problems that primary chicken preadipocytes cannot be passed on indefinitely, heterogeneity, and genetic background differences in cells from different sources, which lead to the difficulty of obtaining stable and reliable results in research, and provides an immortalized chicken preadipocyte Methods

Method used

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  • Method for building immortalized preadipocytes of chicken
  • Method for building immortalized preadipocytes of chicken
  • Method for building immortalized preadipocytes of chicken

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specific Embodiment approach 1

[0045] Specific embodiment one: the present embodiment establishes the method for chicken immortalized preadipocyte to carry out according to the following steps:

[0046] 1. Cloning the full-length coding region sequence of chTERT:

[0047] First, the total RNA of chicken embryo tissue of 4-day-old AA broiler chicken was extracted by RNA extraction kit, and the total RNA of chicken embryo tissue extracted was reverse-transcribed by reverse transcription kit to obtain cDNA of chicken embryo tissue, and three pairs of amplification primers were designed respectively, Using chicken embryo tissue cDNA as a template, the entire coding region sequence chTERT-T1, chTERT-T2 and chTERT-T3 of chTERT were amplified by PCR in three sections, and the amplified products of the three sections were detected by 1% agarose gel electrophoresis and gel electrophoresis. The recovery kit was used for recovery and purification, and PCR amplification primer P7 was designed, and the two fragments of ...

specific Embodiment approach 2

[0123] Embodiment 2: This embodiment is to further illustrate the PCR amplification of chTERT-T1 fragments in step 1 of the method for establishing chicken immortalized preadipocytes described in Embodiment 1. In step 1, PCR amplification of chTERT-T1 The fragment reaction system is 50 μL and consists of the following components:

[0124]

[0125]

[0126] PCR amplification conditions were: denaturation at 95°C for 5 min, denaturation at 95°C for 60 s, annealing at 76.1°C for 25 s, extension at 72°C for 30 s, a total of 30 cycles, extension at 72°C for 5 min, and incubation at 4°C. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0127]Specific embodiment three: This embodiment is to further illustrate the PCR amplification of chTERT-T2 in step one of the method for establishing chicken immortalized preadipocytes described in specific embodiment one, and PCR amplification of the chTERT-T2 fragment in step one The reaction system is 50 μL and consists of the following components:

[0128]

[0129] PCR amplification conditions were: denaturation at 94°C for 5 min, denaturation at 94°C for 45 s, annealing at 63.7°C for 25 s, extension at 72°C for 130 s, a total of 30 cycles, extension at 72°C for 5 min, and incubation at 4°C. Other steps and parameters are the same as those in Embodiment 1 or Embodiment 2.

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Abstract

The invention relates to a method for building the immortalized preadipocytes of chicken, which can be used for solving the problems that the stable and reliable result is difficult to obtain by the research as the preadipocytes of primary chicken can not go down to the future generation infinitely and is heterogeneous, cells with different sources have different genetic backgrounds, and the like. The method comprises the steps of firstly, cloning human chicken telomerase reverse transcriptase (chTERT) genes; secondly, cloning chicken telomerase (chTR) RNA genes; thirdly, constructing a reverse transcribing virus expression vector; fourthly, packaging and preparing reverse transcribing virus; and fifthly, obtaining an immortalized chicken preadipocyte system ICPA-1 and an immortalized chicken preadipocyte system ICPA-2. The method is applied to the field of building the immortalized preadipocytes of the chicken.

Description

technical field [0001] The present invention relates to a method for establishing immortalized preadipocytes of chickens. Background technique [0002] Fat is not only an energy storage tissue, but also an important endocrine organ during the growth and development of the body. Dysfunction of fat can lead to obesity, metabolic syndrome and complex diseases such as diabetes, atherosclerosis, dyslipidemia, hypertension and malignancy. As the incidence of obesity and related diseases is increasing year by year globally, the research on adipocyte differentiation and adipocyte function has become a hot spot in life science research. [0003] In the field of agricultural production, excessive accumulation of abdominal fat is a difficult problem in broiler industry. In order to solve the problem of excessive accumulation of abdominal fat in broiler chickens in actual production and to cultivate high-quality low-fat broiler chickens, we need to carry out targeted research on the d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 王宁王伟李辉闫晓红
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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