Hello, everyone. Recently I'm trying to upregulate a protein in a heterzygous mouse or downregulate this protein in a wildtype mouse to conduct the rescue experiment. Because this gene is longer than 3K bp, I've tried to package lentivirus and AAV, or transient transfection to overexpress this protein, the titration or efficiency were always very low. I prefer not to downregualte it in a wildtype mouse via shRNA cause it has several other interaction partners. If I cannot finish the upregulation, I have to choose to downregulate it. What suggesstions do you have? Thank you so much!
For cell based experiments:
3kb is large for lenti, and will probably have weak expression. If transient expression (3-7 days) is adequate for your experiments, I'd recommend performing in vitro transcription and transfecting your mRNA using an mRNA transfection compound such as MessengerMAX, Trans-IT mRNA, or equivalent. I've found this approach outperforms plasmid or viral-based expression for cDNAs 2kb or larger.
For in vivo (or cellular) experiments:
Overexpression at endogenous loci can be engineered using Cas9 or TALEN constructs fused to transcription activator domains. See PMID 25307933 or 25494202 for some recent progress in this area.
3 kbp is not extraordinarily long. I do not think that you have overexpression problems because of the gene length. It is more likely that the protein is toxic to cells, at least when overexpressed.
Thanks, Andriy. Actually this length already limited the package of the lentivirus in my hands. Maybe you are right, I will choose moderate knockdown in the wildtype mouse, cause at least the heterzygous are quite normal which indicates no obvious side effects happen when it is downregulated.
For cell based experiments:
3kb is large for lenti, and will probably have weak expression. If transient expression (3-7 days) is adequate for your experiments, I'd recommend performing in vitro transcription and transfecting your mRNA using an mRNA transfection compound such as MessengerMAX, Trans-IT mRNA, or equivalent. I've found this approach outperforms plasmid or viral-based expression for cDNAs 2kb or larger.
For in vivo (or cellular) experiments:
Overexpression at endogenous loci can be engineered using Cas9 or TALEN constructs fused to transcription activator domains. See PMID 25307933 or 25494202 for some recent progress in this area.
Dear Daniel, thank you so much! Yes, I am also considering transfect mRNA instead of DNA, because it will theoretically reaches higher efficiency. But I' m not sure the difficulty of the in vitro transcription step. For the in vivo experiments, I will also have a try based on your suggestions.
IVT is fairly easy. The main requirements are a T7 or SP6 promoter 5' to your cDNA and a restriction enzyme site to linearize your DNA template 3' to your stop codon (preferably 10-20 bp downstream).
Thank you, Daniel. I'm working on it now. Hope it will meet my needs. ^_^
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