Gene Delivery Tools Webinar Series (Part 1)은 Lentivirus 소개 및 적용에 대한 웨비나입니다. 바이러스 벡터를 이용한 Gene delivery는 target gene의 전달 효율을 높일 수 있기 때문에 특정 세포주에 관심 있는 유전체를 전달하는 연구용 도구로 많이 사용하고 있습니다.
Part 1: Lentivirus
- Background on viral gene delivery
- Lentivirus 101 (What, How, and Why)
- Safety
- How to produce and use lentivirus
- OriGene solutions & services
Q&A :
Is there any safety issue with this pLenti vector?
The pLenti vector is a third generation lentiviral vector and it is the safest lenti-viral vector because both LTRs are truncated. Please contact the biosafety office at your institution prior to use of the pLenti vector for permission and for further institution-specific instructions. BL2/(+) conditions should be used at all times when handling lentivirus. All decontamination steps should be performed using 70% ethanol/1% SDS. Gloves should be worn at all times when handling lentiviral preparations, transfected cells or the combined transfection reagent and lentiviral DNA.
Do you use any specialized E. coli strain for making plasmids for Lentiviral cloning vectors?
For viral plasmids, we recommend using NEB Stable or Stbl3 competent cells.
After transfection but before lentivirus collection, should you culture the HEK-293T in serum-free media?
No, we use complete growth media: https://cdn.origene.com/assets/documents/lentiviral-packaging-kit/tr30040-product-manual.pdf
The serum-free or reduced serum media are for making the transfection mixture.
I tried to produce viruses recently by for some reason I cannot obtain lentiviral particle. Transfection is OK (as I can see GFP signal) but then I cannot infect cells with the supernatant? What could be the reason why?
The titer might be low. We use a p24 ELISA kit to measure the titer: https://www.origene.com/products/cdna-clones/lentiviral-particles/lenti-titer-p24-elisa-kit
If the titer is low, you can try Lentiviral Concentrator: https://www.origene.com/products/cdna-clones/lentiviral-particles/lenti-concentrator
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