GB/T 38788-2020 in English
VALIDTechnical specification for establishment of porcine pluripotent stem cells
- Issued on:2020-04-28
- Implemented on:2020-11-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
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《GB/T 38788-2020猪多能干细胞建系技术规范》由424-cnis（中国标准化研究院）归口，主管部门为国家市场监督管理总局。
Introduction
Standard Overview and Technical Background
GB/T 38788-2020 is my country's first special technical standard for the establishment of pig pluripotent stem cells, which was formulated by the Ministry of Agriculture and Rural Affairs. This standard is based on the 2012 Nobel Prize in Physiology induced pluripotent stem cell technology breakthrough, combined with my country's scientific research advantages in the field of pig models, filling the gap in the large animal stem cell standard system.
Definition of core terms
| Terms | Definition | Application examples |
|---|---|---|
| Naive stem cells | Pluripotent state with germline chimerism | Can be used to prepare transgenic pig models |
| Primed stem cells | Only retain the ability to differentiate into the three germ layers | Suitable for drug screening platforms |
| Teratoma | Benign tumor of the three germ layer tissues | Gold standard for pluripotency verification |
Key Technical Requirements
5.1 Establishment of Induced Pluripotent Stem Cell Lines
The specification specifies two methods of gene introduction: integrating vectors (retrovirus/lentivirus) and non-integrating vectors (adenovirus/plasmid). Experimental data show that the clone formation rate can reach 0.1-0.5% when using the Episomal system, which is significantly higher than the 0.01% of plasmid transfection.
5.2 Embryonic stem cell isolation
Three methods for obtaining source cells are recommended:
1) Whole embryo inoculation method: Applicable to 4-8 cell stage embryos
2) Mechanical cutting method: Accuracy requirement ±20μm
3) Enzyme digestion method: Trypsin concentration is strictly controlled at 0.25%
Quality Verification System
| Test items | Qualification criteria | Methodology |
|---|---|---|
| Karyotype analysis | 38,XX/XY | G banding technology |
| X chromosome status | XaXa/XaXi | XIST gene detection |
| Pluripotency genes | POU5F1+ NANOG+ | qRT-PCR |
Implementation suggestions
Key points of operation specifications
1. It is recommended to use mouse embryonic fibroblasts treated with mitomycin C as feeder cells
2. The freezing procedure must use a programmed cooling device with a cooling rate of ≤2°C/min
3. The teratoma experiment requires the use of NOD/SCID mouse strain
Common problem handling
Differentiation control: Adding 2i/LIF inhibitor combination can maintain the initial state;
Mycoplasma contamination: It is recommended to use PCR method to test monthly
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