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Frequently asked questions for X-MAN Cell Lines

Jun 10, 2016 2:27:59 PM No Comments

Here you'll find a complete list of our most frequently asked questions relating to X-MAN cell lines. How are they made, how are they validated, and how can you use them? Read on to find out.

For FAQs specific to HAP1 knockout cells click here

What are X-MAN Cell Lines?

Horizon's X-MAN® (Mutant And Normal) cell lines are genetically engineered cell pairs containing defined genomic modifications, providing a model system for the study of the role of genes in cellular processes and drug responses. Cell lines are provided with a matched wild type control parental cell line ensuring phenotypes can be attributed directly to the genotype.

What should I use X-MAN Cell Lines for?

Using Horizon’s X-MAN Cell Lines you can:

  • Study loss or gain of function mutations at the endogenous level
  • Initiate target identification and validation studies
  • Identify responsive or resistant patient populations in a human cell line model
  • Analyse expression, protein interactions and localization using endogenously tagged genes

Are mutant cell lines provided with a wild type control?

Cell lines are provided as isogenic pairs – the modified mutant line and the originating wild type parental line.

How are X-MAN® Cell Lines validated?

All X-MAN Cell Lines are validated by PCR amplification and Sanger Sequencing to confirm the mutation at the genomic level. Cell lines generated using rAAV are also subjected to an additional digital PCR validation step. To enable you to verify our results, we provide our sequencing result, including primer sequences.

Under what conditions should I store my X-MAN Cell Lines?

Our cell lines are shipped on dry ice but should be stored in standard mammalian cell culture storage conditions using liquid nitrogen for long-term storage.

Do X-MAN Cell Lines need to be maintained in selection?

The cells do not need to be maintained in selection as the mutation is permanent, and the cell line is clonal.

How does Horizon confirm X-MAN® cell lines are free from contamination?

We follow strict bio-banking procedures and our cells are regularly tested for contamination, including mycoplasma.

My cells failed to survive thawing, can you provide me with a replacement?

Our Terms and Conditions allow for 1 free of charge replacement vial within two months of receiving the cell line. We recommend the cells are expanded and you freeze stock immediately to avoid being charged for replacement line in the future.

Is there an optimal number of cells I should freeze down per aliquot for future use?

We freeze 1 x 106 cells per vial as our standard practice.

Do you have any references specific for the line I purchased?

If available, a reference for the mutant cell line is provided on the product page.

Why don’t you have functional data/ expression/ other validation data for X-MAN Cell Lines?

All modifications are validated by sequencing at the genomic level, but because every gene, protein and functional readout is different, it is not practical to assess them all. Forgoing functional validation allows us to keep cell line costs down.

We recommend validating the protein expression by Western blot or equivalent.

If you have any concerns about our X-MAN® Cell Lines, please contact us.

Do you provide the PCR conditions for your cell line genomic validation?

The primer sequences used for our internal genomic validation are provided on the Certificate of Analysis and Datasheet provided with the cell lines. We do not provide the PCR reaction conditions as these should be optimized within your own laboratory.

What other validations do you recommend to be performed?

The ultimate proof that a cell line does not express the gene of interest is protein expression analysis by Western blot or similar. Unfortunately, due to the costs and time involved in optimising any new antibody, Horizon cannot test protein expression in all its cell lines. As such we recommend that protein expression is assessed in cell lines prior to proceeding with downstream experiments.

Please contact us if a HAP1 knockout cell line retains gene expression as detected by Western blotting.

The cell morphology and growth rate is different between the mutant cell line and the parental?

We do not monitor the morphology or growth rates of the cells during the gene editing and banking so we cannot provide in-house data for these parameters. If you would like advice, please contact us and our Technical Support team would be happy to discuss your findings with the in-house scientists.

I performed a Western Blot, and observed no difference in the expression between the knockout cell line and the parental cell line?

We request that you send an image of the Western blot along with details of your experimental design to technical@horizondiscovery.com for evaluation. We can then arrange for the shipment of an alternative clone if available or restart the gene engineering to target a different exon of the same gene.

I treated the cells with an inhibitor/drug/etc. and did not observe the phenotypic difference I expected between the parental and the mutant cell line?

With all of the cell lines, we carry out genomic validation via sequencing/PCR to confirm the presence of the mutation. We do not perform functional validation.

In this case please contact technical@horizondiscovery.com and we may be able to suggest a clone in another cell line.

What’s the difference between "on demand" and "off-the-shelf" cell lines?

Off the Shelf means that we have those cell lines for that specific gene already made, whereas On Demand requires us to make the cell line for a specific gene of interest.

The Off the Shelf can be supplied in 1-2 weeks, whereas the On Demand is about 12 weeks because we must generate the knockout cell line using CRISPR-Cas9. If you require a modification made in a non-haploid cell line please refer to our Custom Cell Line Engineering page

Still have an outstanding question?

Contact our team

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