Method for constructing OX40 gene modified humanized animal model and application thereof
A technology of genetic modification and animal models, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve problems such as unrealized exon humanization, to ensure success rate, reduce The effect of gene transcription
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Example Embodiment
[0050] Example 1: Preparation of Guide RNA, Cas9mRNA and Donor vector
[0051] 1. Guide RNA (sgRNA)
[0052] 1. sgRNA sequence
[0053] Determine the most suitable sgRNA sequence through multiple experiments:
[0054] ox40-S4 sgRNA:cccacagcccttctgctgct ggg
[0055] ox40-S8 sgRNA: cacagacgcacactttactc tgg
[0056] 2. sgRNA construction
[0057] (1) Preparation: Bsa I linearized pUC57-gRNA-T7 vector and dephosphorylated it with CIAP (TaKaRa, Cat#2250A).
[0058] (2) Annealing: The upstream and downstream primers are diluted to 100umol in a ratio of 1:1. Anneal at 95°C for 5 min. (Go to hot cover)
[0059] (3) Phosphoric acid addition: 2ul of the annealed product is treated with phosphoric acid (TaKaRa, Cat#2021S), the system is as follows:
[0060]
[0061] Thermal cycler to remove the lid, program: 37°C for 30 min.
[0062] (4) Connection
[0063] The product after adding phosphoric acid was connected to pUC57-gDNA-T7 linearized and dephosphorylated.
[0064] system:
[0065] pUC57-gDNA-T7 ...
Example Embodiment
[0187] Example 2 Injection sample preparation
[0188] 1. First write the label on the centrifuge tube according to the name of the injection table, and add it to the tube in order according to the volume on the pre-calculated preparation table:
[0189] Injection buffer→Cas9-mRNA→Donor→sgRNA.
[0190] 2. After the sample is mixed, close the lid tightly and flick the EP tube 8-10 times with your fingers to mix it evenly.
[0191] 3. Centrifuge the mixed sample at 12000g for 5 minutes in a pre-cooled centrifuge.
[0192] Injection sample preparation (total volume 50ul):
[0193] Cas9: 100ng / ul
[0194] Donor:100ng / ul
[0195] sgRNA: 20ng / ul each.
Example Embodiment
[0196] Example 3 Injection and transplantation
[0197] 1. IVF provides embryos for injection
[0198] 1) Material:
[0199] Female rat: 3-4 weeks old, 13-15g
[0200] Male rat: 3-6 months, more than 10 days alone
[0201] Human chorionic gonadotropin (HCG) (Ningbo Sansheng Pharmaceutical Co., Ltd.)
[0202] Maternal Serum Gonadotropin (PMSG) (Ningbo Sansheng Pharmaceutical Co., Ltd.)
[0203] Mineral oil (Sigma M5310)
[0204] HTF (use Sigma reagent to prepare)
[0205] c-TYH (use Sigma reagent to prepare)
[0206] 2) Superovulation: intraperitoneal injection
[0207] Day1: The oocytes were injected with PMSG (Ningbo Sansheng Pharmaceutical Co., Ltd.) 5IU / mouse from 8:30-9:00 in the morning.
[0208] Day3: Donor oocytes were injected with HCG (Ningbo Sansheng Pharmaceutical Co., Ltd.) 5IU / mouse at 8:15-8:45 in the morning.
[0209] 3) Sperm collection and vitality test
[0210] Collection time: Sperm collection between 22:00-22:05
[0211] 4) Collect eggs
[0212] After 13-14 hours of HCG injecti...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap