hey, I would like to ask how to construct a stable cell line which can produce Reelin?
Like in normal way RNA-cDNA-Molecular Cloning-transfection ? or there is other method, cuz this mRNA length looks quite long about 10kd.....
Hi Lyla Kou,
If u have access to BSL2 laboratory, you can use lentiviral systems.
For stable cell line production the lentiviral systems are one approach. But you can use adenoviral systems also. Other approaches exist also. I have some experience in lentivirus (3rd generation) propagation and transduction. If you need any help just feel free to write! I have got detailed protocols for the entire process (from transduction to clone screening).
On Addgene site you can find the Lentiviral Guide (https://www.addgene.org/viral-vectors/lentivirus/lenti-guide/). It's very useful and easy to follow. This guide is good from the basics to the advanced level.
Here you can read an another very good paper about the lentiviral systemArticle Nucleic Acid Delivery: Lentiviral and Retroviral Vectors
Ther are 2nd and 3rd generation systems. The 3rd genetartion system is more safer. All of the required plasmids are available from Addgene site (https://www.addgene.org/) also.
After the co-transfection of the virus propagating host cell line (e.g. HEK 293 or HEK 293-AD) with the 3 plasmids then lentiviruses had assembled in host cells and the ready viruses has escaped into the cell culture media.
You can use the supernatant of this cell line without concentration also. But i suggest to split the SN into smaller single use aliquot followed by freeze at -80°C. Avoid frequenty freez-thaw cycles. Becuse every cycle can decrease the virus titer by 25 %.
You should concentrate your lentivirus. No need expensive equipment for virus islation neither if you use the PEG precipitation protocol despite of ultracentrifugation.
I have been used Reverse Transcriptase Assay to check virus titer. I havent't used the p24 Antigen Assay which is very common. Here you can read a paper about titration method comparison.Article Comparison of lentiviral vector titration methods
Bests,
Tamas
I guess you have to use "the classic way". Lenti/Retroviruses do not have the packaging capacity to handle 10 kbp. So RNA-cDNA-Molecular Cloning-transient transfection-selection-clone screening. We do have decent experience with Q5 polymerase for amplification of such long targets, so it is definately possible. At least, if your cDNA is covering the whole transcript (use RandHex and Oligo-dT both). There are other viral systems without such a packaging limit, but I am not experienced with them and thus cannot help you there. Plus, they might be BSL2.
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