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Sheep induced pluripotent stem cell and preparation method thereof

A technology of pluripotent stem cells and stem cells, applied in the field of induced pluripotent stem cells and their preparation

Inactive Publication Date: 2012-07-18
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since iPS cells are not 100% ES cells, whether they can be applied to species that cannot establish ES cells using traditional methods is still unknown, and there is no report in the prior art.”

Method used

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  • Sheep induced pluripotent stem cell and preparation method thereof
  • Sheep induced pluripotent stem cell and preparation method thereof
  • Sheep induced pluripotent stem cell and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, Construction of lentiviral vector

[0039] 1.1. From the NCBI website (http: / / www.ncbi.nlm.nih.gov / ), query the specific expression or high expression of specific genes (Oct4, Sox2, c-Myc, Klf4, Lin28, Nanog, SV40, hTERT ), primers were designed according to the sequence of the coding region, and restriction sites were introduced. The primer sequences are shown in Table 1, wherein F represents the forward primer, and R represents the reverse primer.

[0040] Table 1 Primer sequences

[0041]

[0042] Note: The uppercase letters in the primer sequences are the restriction restriction sites introduced.

[0043] 1.2. PCR amplification

[0044] Using the total human cDNA as a template, PCR amplification was performed using the primers of each gene in Table 1, as follows:

[0045] Reaction system (25μl): 10×pfxMix 2.5μl, AccuPrime pfx enzyme 0.2μl, upstream and downstream primers (50μM) each 0.25μl, template 0.25μl, ddH 2 O 21.55 μl.

[0046] Reaction c...

Embodiment 2

[0050] Embodiment 2, cell culture

[0051] 2.1. Culture of sheep primary ear tip fibroblasts (PEF)

[0052] Take sheep ears, wash with 75% alcohol and shave, soak in PBS containing double antibodies (penicillin, streptomycin) for 15 minutes, then use PBS and serum-free medium (D-MEM) to wash the ears several times, and then put The ear was soaked in a small amount of D-MEM containing 10% 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 10% 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,...

Embodiment 3

[0056] Embodiment 3, virus packaging

[0057] 3.1. Amplification of packaging plasmid

[0058] Eight kinds of correctly identified vectors obtained in Example 1 were transformed into competent bacteria to be amplified, and AxygenAxyPrep 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.

[0059] 3.2. Transfection

[0060] According to Invitrogen transfection kit (ViraPower TM Lentiviral Expression Systems), using the transfection reagent Lipofectamine TM In 2000, the...

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Abstract

The invention relates to a sheep induced pluripotent stem cell and a preparation method thereof. The preparation method comprises the steps of: A) constructing lentivirus vectors with transcription factors, wherein the transcription factors are selected from Oct4, Sox2, c-Myc, K1f4, Lin28, Nanog, SV40 and hTERT; and B) infecting sheep adult cells to the transcription factors in a combining form by adopting the lentivirus vectors obtained in the step A), selecting cloned passages with forms similar to those of embryonic stem cells for culturing, and sieving the cells satisfied with characteristics of embryonic stem cells for cloning so as to obtain the sheep induced pluripotent stem cells. The sheep induced pluripotent stem cell and the preparation method thereof, disclosed by the invention, are good for determining most suitable culture condition and method of sheep ES cell establishment system; the sheep induced pluripotent stem cell is a good carrier for sheep gene targeting; and the sheep induced pluripotent stem cell is beneficial to revealing gene functions and complex development events of sheep and can be used for improving species and promoting economic growth.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an induced pluripotent stem cell (Induced pluripotent stem cell, iPS cell for short) which reprograms a sheep adult cell into a similar embryonic stem cell and a preparation method thereof. Background technique [0002] Sheep are large artiodactyla animals, whose genome and organ size are similar to those of humans, and are widely used in animal husbandry and agriculture, and can be used to create human disease models; use their organs as donors for human organ transplantation; use transgenic technology Manufacturing pharmaceutical proteins; improving species and more. [0003] Embryonic stem cells are derived from blastocysts, which can multiply indefinitely while maintaining pluripotency, that is, a type of progenitor cell that can differentiate into three germ layers. The reverse genetics method based on embryonic stem cell research has a huge role in promoting ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N15/867C12N5/10
Inventor 肖磊鲍磊何丽夏子
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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