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Frequently Asked Questions For HAP1 Knockout Cell Lines

Jun 10, 2016 2:29:00 PM 1 Comment
Here you'll find a complete list of all our most frequently asked questions relating to HAP1 knockout cell lines. If you want to know how they're generated, how they're validated or how to find out if they're right for you - this is a great place to start.

How are Hap1 knockout cell lines generated?

HAP1 knockouts are engineered using the CRISPR-Cas9 system to introduce frameshift mutations into the coding sequence of genes of interest.

Can I use the HAP1 cell line for my research?

The HAP1 cell line has been applied across a wide range of biological processes, such as DNA damage repair pathway and stress responses, as well as in disease modeling. These HAP1 review articles show the broad applicability of the HAP1 cell line, and provide characterization data to help your research.

What type of cells are HAP1 cells?

HAP1 is a near-haploid human cell line that was derived from the male chronic myelogenous leukemia (CML) cell line KBM-7 (see Carette et al. Nature. 2011.). HAP1 cells are adherent with fibroblast-like morphology.

View our overview of HAP1 cells for more information.

How are HAP1 knockouts validated?

All HAP1 knockout cell lines are validated by PCR amplification and Sanger Sequencing to confirm the mutation at the genomic level. To enable you to verify our results, we provide our sequencing result, including primer sequences.

Further to this HAP1 wild-type cells have been extensively characterized by whole genome sequencing and transcriptome analysis.

How do you culture HAP1 cells?

HAP1 cells are grown in Iscove’s Modified Dulbecco’s Medium (IMDM) in the presence of 10% fetal calf serum and penicillin/ streptomycin. Cells are passaged every 48h at a dilution of 1:10 or 1:20, depending on the initial density. More detailed information is available here. Additionally, this information is listed on the Certificate of Analysis for the cell line.

What will I receive when I order a HAP1 knockout cell line?

Customers obtain a clonal HAP1 cell line, bearing a frameshift mutation in an exon of the gene of interest. Typically, the mutation is placed closed to the 5’end of the gene, ie. in exon 1 or 2.

The mutation is described in the clone datasheet enclosed upon shipment and can be verified by a PCR on genomic DNA followed by Sanger sequencing.

All mutant cell lines are provided as an isogenic pair with the wild type line. This provides a control for your downstream experiments

Do HAP1 cell lines contain antibiotic resistance cassettes?

The HAP1 Knockout cell lines do not contain antibiotic resistance cassettes. The guide RNAs used to generate the knockouts have a Blasticidine resistance gene which enables enrichment of transfected cells, but this is only expressed transiently and will be lost during culture.

How do you know that HAP1 knockout cells don’t contain off-target modifications?

While we cannot exclude entirely the possibility of off target modifications in addition to on target cleavage, we believe it represents a relatively low and controllable risk, as a number of recent publications have demonstrated that the CRISPR-Cas9 system is to be highly specific (e.g. Cencic et al).

Further to this a collaborator group have published a paper where they examine off-targets in the HAP1 cell line and observe very low frequencies.

At Horizon, to mitigate the risk still further our in-house selection algorithms warrant that only those guide RNAs with minimal predicted off-target sites in the human genome are chosen. Further to this, scientists can control their experiments through the use of multiple, independent clones or rescue of the knockout with a wild-type cDNA.

Do you have any data on additional endogenous mutations in the HAP1 cell line?

We guarantee only the genomic validation that is provided for the listed mutations in the certificate of analysis. However, we have subjected HAP1 cells to whole-genome sequencing and published these data (Essletzbichler P. et al., Genome Res. 2014)

I have never worked with HAP1 cells before. How do I know if they look correct?

Our Technical Support team is happy to provide a visual inspection of your culture if you send an image to technical@horizondiscovery.com. In addition, further information and example images can be found in our HAP1 Cell Culture Guidelines.

Are the HAP1 cell lines complete knockouts?

The clonal cell line you obtain bears a frameshift mutation in your gene of interest. For this reason, we do not expect any residual gene expression of the wild-type allele. However, there are two rare scenarios in which such expression of the wild-type allele may still be detectable:

  1. There is an alternative start codon downstream of the actual ATG.
  2. There is an alternatively spliced transcript that does not include the edited exon.

If you detect residual gene expression in one of our clones, please contact us

Can I verify the knockout by qRT-PCR?

Knockouts of genes in HAP1 cell lines is achieved through the introduction of small indels that give rise to frameshift mutations. While these can render the coding sequence non-functional, this non-sense transcript can in some cases still be expressed, leading to misleading qRT-PCR results.

As such, we strongly recommend validation at the protein level.

Are the HAP1 cells still haploid after gene knockout?

The majority of clones are haploid by the end of KO generation, but we do not provide verification of ploidy. However, if the mutant cell line becomes diploid, this will still be a full knockout.

If your experiments require a haploid clone, please contact us.

Can you please provide some references for HAP1 cells?

Selected publications that describe HAP1 cells include:

Is my gene of interest expressed in HAP1 cells?

We have analysed gene expression in HAP1 cells by RNAseq. Please contact us with your gene of interest and we will be able to tell you whether it is expressed.

For how many passages can HAP1 cells be expected to grow?

In our experience, the HAP1 cells can be maintained up to 20 passages if the cell culture guidelines are followed.

What’s the difference between "on demand" and "off-the-shelf" HAP1 cell lines?

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

Do HAP1 cells respond to TNFα induction?

We have observed a response in HAP1 cells to both TNFα and IFNγ. If you have another pathway you’re interested in please contact us.

Do you have a protocol for HAP1 transfection?

We transfect our HAP1 cell lines on a routine basis by Lipofection, using Turbofectin from OriGene (http://www.origene.com/cdna/turbofectin.mspx) and OptiMem from Life Technologies. We seed 800.00 cells in a 6-well one day before transfection. The transfection efficiency is around 25%.

We have received feedback from our customers that transfection of the HAP1 cell line is also achievable with electroporation. For this protocol, a Neon Electroporation system at 1575V, 10 ms, 3 pulses was found to be most useful.

Why are there multiple HAP1 cell lines listed for my gene of interest?

When we perform the CRISPR gene editing, we often isolate multiple clones with successful frameshifts. The differences between them is often the number of bases that are inserted or deleted.

If you click on the product pages for each of the clones and scroll down the page, there is a pdf link with the datasheets attached. This describes the location of the frameshift and you can then determine which is more suitable for your needs.

Has the genome of HAP1 cells been sequenced? Is this data available?

Yes – the genome of HAP1 cells was sequenced in both Kim et al and Essletzbichler et al. You may also find the sequencing data for the latter useful.

How big are HAP1 cells?

HAP1 cells are small compared to other cell lines, at around 11microm in diameter.

Can HAP1 cells be used in xenografts?

Tumorigenicity of HAP1 cells in immuno-compromised mice has been assessed by OncoTest.

Three groups with three female NMRI nu/nu mice each received bilaterial subcutaneous injections (left and right flank) of HAP-1 cells supplied by Haplogen Genomics GmbH (5x105, 1x106, 5x106 cells per injection site).

During the 90-day observation phase tumors grew out at 8 of 18 injection sites in 6 out of 9 mice. Their volumes ranged from 111 mm3 to larger than 1700 mm3. The latency period for the onset of tumor growth ranged from 19 to 78 days. No deaths or progressive body weight loss of tumor-bearing mice and no clinical signs were observed.

Are HAP1 cell lines genetically stable (i.e. do they stay haploid)?

We have observed that haploid cell lines will spontaneously diploidise over time. If a gene has been knocked out, diploidisation will not affect knockout status, but to minimise the potential impact of diploidisation one of the following approaches is recommended:

  • Work with cells at very low passage. Prepare stocks of low passage cells and revive frequently.
  • Culture knockout and wild type cells in parallel and allow genomes to stabilise. Then use these high passage cells for experimental analyses.

Why are the cells haploid?

HAP1 cells are derived from a chronic myelogenous leukemia (CML), a neoplastic disease in which near-haploidy, while rare, is more frequently observed.

How do I know if HAP1 cells are suitable for my studying a particular biological pathway?

Please contact us to discuss your experimental needs – internal research projects with this cell line are ongoing and we may have data we can share.


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