Q: Do you guarantee gene knockdown with Lentiviral shRNA gene knockdown/verification kit?

Yes. We guarantee at least one of the three shRNAs will trigger >80% mRNA or protein knockdown.

Q: Which promoter should I use for driving shRNA?

Promoter activities varies in different cell lines, which may affect the expression level of the shRNA and the level of gene knockdown. If you do not know which promoter works best for your cells, we suggest to use our Promoter selection plate to figure out which promoter to choose.

Q: Do you provide tissue-specific promoter driving shRNA?

We have lentiviral cloning vectors with tissue-specific promoter for driving shRNA expression, will be happy to generate custom shRNA.

Q: How do you titrate shRNA lentivirus?

We do functional titration on HT1080 cells with series dilutions of the purified lentivirus and count puromycin resistant cell clones.

Q: What MOI should I use for transducing my cells?

Although lentivirus can transduce almost all kinds of cells, the transduction efficiency varies a lot in different cell lines. We suggest doing a pilot experiment with our premade fluorescent lentivirus to set up the transduction condition before the real experiments.

Q: Where can I find shRNA sequence?

The shRNA sequences will be on the document shipping with the products.  You can always request the information by contacting us.

Q: What is the biosafety level for using lentivirus?

Biosafety level 2.

Q: Can I use FenicsBIO’s premade lentivirus for in vivo experiments?

Yes. The premade lentivirus is concentrated and purified. The titer and purity are high enough for in vivo use.

Q: How do you determine lentivirus titer?

We do functional titration on mammalian cells using series dilutions of the lentivirus. If there is antibiotic selection marker on the lentiviral construct, the titer is determined by antibiotic resistant cell clones. If there is fluorescent reporter on the lentiviral construct, the titer is determined by counting fluorescent positive cell clones. If there is no marker on the lentiviral construct, the titer is determined by real-time PCR with prepared genomic DNA from transduced mammalian cells.

Q: What MOI should I use for my cells?

Although lentivirus can transduce almost all kinds of cells, the transduction efficiency varies a lot in different cell lines. We suggest doing a pilot experiment with our premade fluorescent lentivirus to set up the transduction condition before the real experiments.

Q: Which generation is FenicsBio’s lentiviral vector?

Our premade lentiviruses are produced from third generation lentiviral vectors, with a chimeric 5’LTR and truncated 3’LTR.

Q: How were FenicsBIO’s premade stable cell lines generated?

All the premade stable cell lines were generated using our premade lentiviruses. After transducing the parental cells, antibiotic selection was used to select the transduction positive cells.

Q: Are FenicsBIO’s premade stable cell lines pooled stable cells or clonal stable cells?

All the cell lines are pooled stable cells. If you need clonal stable cells, we would be happy to custom generate the stable clones for you.

Q: What biosafety level are required for handling FenicsBIO’s premade stable cells?

All the stable cells were generated using lentiviral particles. Although the parental cells are only on biosafety level I, all the stable cells should be handled with biosafety level 2 protocol.

Q: What are in the sgRNA gene knockout kit?

The sgRNA gene knockout kit includes lentiviral particles for a set of three sgRNAs mixture, and a pair of Indel screening PCR primers.

Q: Where can I find sgRNA and PAM sequences?

The sequences will be on the data sheet shipped with the products. You can always contact us for the sequences.

Q: What is the advantage of using three sgRNAs?

Using multiple sgRNAs has been shown to dramatically increase gene knockout efficiency. FenicsBIO’s bioinformatics team designed a three sgRNAs strategy that deletes a large fragment in the target gene.

Q: How do I know my cell line knockout experiment is successful?

With FenicsBIO’s Indel screening primers, you can estimate the knockout efficiency by comparing PCR products from genomic DNA prepared from pooled transduced cells and parental cells. The knockout cells will show a shorter PCR product as predicted. This confirmation will save you lots of time before generating single cell clones.

Q: How do I verify gene knockout cell lines?

We suggest screening the candidate cell clones using provided Indel screening primers first, then send the genomic DNA with shorter PCR products for sequencing verification. If you have an antibody for the target protein, we suggest doing Western blot to verify at the protein level.

Q: How did you verify the knockout cell lines?

All FenicsBIO’s knockout cell lines are sequence verified.

Q: Besides HEK293 and A549 cells, do you provide other knockout cell lines?

For on-shelf products, we only provide HEK293 and A549 knockout cells as shelf products for now. We would be happy to generate the knockout cell line you need as a custom project. Please contact us for a quote.  

Q: What biosafety level is required for handling FenicsBio’s knockout cell lines?

FenicsBio’s knockout cell lines were generated using lentiviral particles. Biosafety level 2 is required for handling these cells.