I am a novice in molecular cloning, and recently I was given a new project to overexpress lncRNA in HEK cells. I am looking for suggestion and guidance for the following queries:
1. On what basis should I select the plasmid for overexpression?
2. The length of the gene is 4.2kb. Do I need to split the gene in 3 or 4 parts and perform cloning?
3. Does the size matters for over expression?
Thank you in advance!
Sweta Gupta
Hi Sweta,
I am planning to do a similar overexpression with a different lncRNA, therefore I was wondering could you find any answers to your posted question regarding this.
Kind regards,
Nazanin
Hi Sweta,
We recently published a paper that well answer your concerns and offer a nearly perfect tool for stable overexpression of lncRNAs. In this study, we found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~2 kb to the 3’-end. Consequently, the secondary structures were changed, the RNA-protein interactions were blocked, and the functions were impaired in certain lncRNAs. To solve this problem, we developed a novel lncRNA expression tool called Expression of LncRNAs with Endogenous Characteristics using Transposon System (ELECTS). By inserting termination signal after the lncRNA sequence, ELECTS produces transcripts without 3'-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, there is no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, efficient and time-saving delivery tool for stable expressing lncRNAs.
The open accession of the full article is available at : https://jnanobiotechnology.biomedcentral.com/articles/10.1186/s12951-021-01044-7
We welcome all the researchers to use our tools. If you have any questions, please feel free to contact me, my email is: [email protected].
- Badges
- Science topic
- Similar topics
- Correction