Hello, Does any one faced transgene silencing after transfection? I have been trying to get good expression from a recombinant antibody molecules cloned in pUC based vector and transfected in CHO cells. Although the cells were selected and checked for expression the expression is very less and there is no reporter gene available to check the transfection efficiency.
I have recently come across the insulation of the transgene on either side with insulator sequences. so did anyone tried the same? If so can i get any protocol or any paper link to learn how to include insulators for achieving high level of transgene expression?
Thanks in advance.
You can target your vector (assisted with CRISPR/TALEN) to safe harbour sites like AAVS1 or the ROSA locus, they are resistant to silencing.
Vasanth Thamodaran I have come across few papers that safe harbor locus for CHO cells is H11. I read about these and will these be the best choice for high level gene expression?
Hi Anand, I am not sure about CHO cells. However, we have tested in human iPSCs, where gene expression using lentivirus vectors has silencing issues and heterogeneity in expression. But, when we express genes from AAVS1 safe harbour site the gene expression is uniform and its maintained for several generations.
Vasanth Thamodaran Thats encouraging and thanks a lot for the insight.
Vasanth Thamodaran how do you insert your gene in safe locus locus? Do you use crispr? if yes can you let me know whether you are designing your own protocols or you are taking help of any kit?
I may echo with Vasanth Thamodaran, as it may happen. In case the only way is to use dCas9.VP4 to reactivate the gene in next generation using promoter or enhancer specific gRNA. Provided that lentiviral vector should have GFP or YFP to sort those cells. Whenever you thaw those preserved cells earlier showed expression, just transfect again to ensure that desirable transgene would express.
Hello Anand, you can get the gene editing tools for targeting the AAVS1 plasmids from addgene (we got it from there). But they are for human AAVS1 region, but still you can check it out if these plasmids can be compatible for the Cho cells (may be if the locus is conserved). In general ,for targeting, you can get either TALEN plasmids (two sets of plasmids pzt-aavs1-R1 and pzt-aavs1-L1) or a sgRNA expressing plasmid that targets the AAVS1 gene (pX330-cripr-T2 plasmid). All these plasmids are available in addgene. For the donor plasmid that has homology arm for the AAVS1 you can get the pAAVS1-cag-cop-egfp plasmid (again available in addgene) and you can replace the egfp gene with your gene of interest and target the construct to the AAVS1 locus. If suppose these talen/crispr tools are not compatible for the CHO genome, you can design the cas9-sgRNA by uploading the AAVS1 sequence of CHO line in the synthego sgRNA design tool or the broad institute sgRNA design tool (available online) clone the oligos in an empty px330-crispr plasmid (addgene) and then target the AAVS1 locus...
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