Preparation method of inducible pluripotent stem cell of goat

A pluripotent stem cell and stem cell technology, applied in the field of preparation of induced pluripotent stem cells, can solve the problems of not finding a culture medium, not obtaining a cell line, restricting the application of transgenic goats and cloned sheep, etc.

Active Publication Date: 2014-12-10
苏州中科细胞转化研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For most other mammals, including pigs, cattle, sheep, etc., although some scientists have tried to establish corresponding embryonic stem cell lines, they have not obtained a recognized cell line. One of the most important reasons is that they have not found ESCs suitable for these animals. totipotent maintenance medium
In addition, the embryonic development process of goats and the pathways for maintaining the totipotency of goat ESCs are not clear enough. As a result, no goat ESC cell lines have been produced so far, which restricts the application of transgenic goats and cloned sheep. From the above, the establishment of goat ES cell lines imminent

Method used

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  • Preparation method of inducible pluripotent stem cell of goat
  • Preparation method of inducible pluripotent stem cell of goat
  • Preparation method of inducible pluripotent stem cell of goat

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, Construction of lentiviral vector

[0046] 1.1. Query specific genes (Oct4, Sox2, c-Myc, Klf4, Lin28, Nanog, hTert, SV40largeT) specifically expressed or highly expressed in stem cells from the NCBI website (http: / / www.ncbi.nlm.nih.gov / ) Antigen and rtTA) coding regions, primers were designed according to the coding region sequences, and restriction sites were introduced, the primer sequences are shown in Table 1 (wherein F represents the forward primer, R represents the reverse primer).

[0047] Table 1 Primer sequence list

[0048]

[0049] Note: The uppercase letters in the primer sequences are the restriction restriction sites introduced.

[0050] 1.2. PCR amplification

[0051] Using the total human cDNA as a template, PCR amplification was performed using the primers of each gene in Table 1, as follows:

[0052] Reaction system (25 μl): 2.5 μl of 10×pfxMix, 0.2 μl of AccuPrime pfx enzyme, 0.25 μl of upstream and downstream primers (50 μM), 0....

Embodiment 2

[0057] Embodiment 2, cell culture

[0058] 2.1. Culture of goat primary ear tip fibroblasts (PEF)

[0059] Take goat ears, wash with 75% alcohol and shave, soak in PBS containing double antibodies (penicillin, streptomycin) for 15min, then use PBS, serum-free medium (D-MEM) to wash the ears several times, and then The ear was soaked in a small amount of D-MEM containing 30% FBS, and at the same time, it was cut into small pieces with sterile scissors, and moved to a culture bottle, keeping a small distance between the small pieces, and the culture bottle was turned upside down. After 6-8 hours, add D-MEM containing 30% FBS, place it upright, and then add a small amount of this medium every day. Generally, fibroblasts can be clearly observed after 3 or 4 days. Passage after about a week, use when passage The cells were washed twice with PBS, digested with 0.25% trypsin at 37°C for 5 min, and terminated with 10% FBS in D-MEM by pipetting. For the first passage, pass 1 to 1 o...

Embodiment 3

[0063] Embodiment 3, virus packaging

[0064] 3.1. Amplification of packaging plasmid

[0065] Nine kinds of correctly identified vectors obtained in Example 1 were transformed into competent bacteria to amplify, and Axygen AxyPrep TM After pumping in the plasma Maxiprep Kit kit (Axygen Company), purify in an ultra-clean workbench, that is, mix with 1 / 10 volume of 3M NaAC and 2 times the volume of absolute ethanol of the plasmid after pumping, and then centrifuge at 13000rpm After 15 minutes, the supernatant was removed, rinsed with 75% ethanol, sucked off the supernatant, and dried in an ultra-clean workbench. The plasmid was dissolved in sterile deionized distilled water, and finally the concentration of the plasmid was determined using a spectrophotometer and gel electrophoresis.

[0066] 3.2. Transfection

[0067] According to Invitrogen transfection kit (ViraPower TM Lentiviral Expression Systems), using the transfection reagent Lipofectamine TM In 2000, the six ...

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Abstract

The invention relates to a preparation method of an inducible pluripotent stem cell of a goat. The preparation method comprises the following steps: A) constructing a lentiviral vector carrying a transcription factor which is selected from Oct4, Sox2, c-Myc, Klf4, Lin28 and Nanog; and B) infecting a goat adult cell by combining the transcription factor with the lentiviral vector prepared in the step A, selecting clone with a shape similar to an embryonic stem cell for subculturing, and preparing the inducible pluripotent stem cell of the goat by screening cell clone in accordance with the characteristics of the embryonic stem cell. According to the preparation method, an optimal culture condition and method of a goat ES (embryonic stem) cell line construction can be established; the inducible pluripotent stem cell of the goat is an excellent vector for goat gene targeting, and the inducible pluripotent stem cell of the goat facilitates the revelation of each gene function and the complex development of the goat.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for preparing goats from adult cells reprogrammed into inducible pluripotent stem cells (Induced pluripotent stem cells, iPS cells for short) similar to embryonic stem cells. Background technique [0002] Goats have great application prospects in animal husbandry and medicine. By operating goat embryonic stem cell (ESC) lines, cloned animals or transgenic animals can be produced, which is of great significance to the construction of human disease models, organ transplantation, improved species, and increased economic benefits. major. [0003] ESCs refer to a class of cells derived from the inner cell mass of mammalian blastocysts. This type of cells is round, with a large nuclear-to-cytoplasmic ratio, good self-stability, unlimited proliferation and self-renewal capabilities, and can be permanently passaged in an in vitro culture environment and maintain a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/071
Inventor 肖磊任江涛朴永俊何丽夏子鲍磊崔春廖静
Owner 苏州中科细胞转化研究院
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