Frequently asked questions about HAP1 cell origins

Our HAP1 knockout cell lines offer a solution to study most genes and can help elucidate a gene's function without the time and screening needed to produce your own cell line from a single clone. Our HAP1 cells can be used in quick proof of concept experiments, assist with antibody validation, or answer questions relating to if a knockout cell line of a particular gene will produce desired data.

To help you decide if the HAP1s are right for your assay, this article will go through common questions our Scientific Support team receives on the cells, including:

• The origin of the HAP1 cells
• How our knockouts are made
• The quality control standards for these products

What is the benefit of a knockout produced in a haploid cell line?

Most normal human cells are diploid; they have two sets of chromosomes. Cancer cell lines and immortalized cultured cell lines often have additional copies of genes. When analyzing a cell line in which to perform a knockout, it may be realized that most cell lines in culture are polyploid. This means there are many copies of the gene of interest due to whole chromosome duplication, or translocations that occur during disease progression, cell line immortalization, or continuous cell culture. To obtain a functional knockout in diploid or polyploid cell lines, one would need to mutate every copy of the gene within the cells. When making a knockout using CRISPR, the different copies of the gene often have different mutations at the DNA level which can give different RNA transcripts and potentially different phenotypes in the cell.

The HAP1 cell line is near haploid meaning that it has, for the most part, one copy of a gene transcribed from a single allele. Since only one allele is expressed, only one allele needs to be knocked out. There are no additional copies of the gene which can mask the knockout or have a different phenotype. This makes HAP1 cells a useful tool for studying gene function, saving time in getting the knockout to you, and makes your results easier to interpret.

What are HAP1 cells? Is my gene haploid in these cells?

The HAP1 cell line originated from KBM-7 (Catalog #C628), a suspension cell line that was derived from cells taken from a myeloid leukemia patient (Andersson, B et al Cancer Genet. Cytogenet 1987, Kontecki et al 1999 Exp Cell Res). KBM-7 cells are near- haploid except for two copies of Chromosome (Chr) 8, and a small section of Chr 15 that is duplicated in Chr 19.

Table 1 Differences between the parental cell types: *C859 has additional sorting on these cells to remove any cells that have diploidized spontaneously and are best used for screening applications.

As these cells are from a myeloid lineage, the KBM-7s have a classic marker of myeloid leukemia cells—the Philadelphia chromosome ( Kotecki et al 1999 Exp Cell Res, Carette et al 2009 in Science).

KBM-7 cells were transfected with Yamanaka factors (Oct4, cMyc, Sox2, and Klf4) to induce pluripotency. Although pluripotency was not achieved, cells transfected with the Yamanaka factors grew out a population of adherent fibroblast-like cells. The HAP1 cell line was obtained from clonal populations of these cells.

HAP1 cells are different from KBM-7 cells in that the HAP1 cells are no longer diploid for Chr 8, but they retain the fragment of Chr 15 in Chr 19 (see Table 1). The fragment of Chr 15 that is duplicated is approximately 30 Mb and contains 330 genes. We use this HAP1 parental cell line to make our knockout cell lines, so, if your gene of interest is in Chr 15, contact Scientific Support and we can let you know if your gene is in this duplication.

What is the difference between the different parental (wildtype) HAP1 cell lines that are offered?

We offer three different parental lines for the HAP1 cells. With the purchase of any of our HAP1 knockout clones, a free parental line is included (Catalog C631). However, we have two additional parental cell lines for purchase.

We offer Screening ready HAP1 cells (C859) which are the same as C631, but we have done some additional sorting on these cells to remove any cells that have diploidized spontaneously. In a quest to obtain a truly haploid model we generated the E-Hap (C669) cell line by removing the duplication of chromosome 15 in chromosome 19 by using CRISPR-Cas9 (Carette et all 2011 Nature). This cell line does still have the Philadelphia chromosome.(Table 1)

Do HAP1 cells express my gene of interest?

HAP1 cells have been characterized by RNAseq analysis. Because of this characterization, the relative expression of each of the genes in this cell line is known in basal, unstimulated conditions. On each HAP1 gene-specific product page we have included the transcripts per million (TPM) from this analysis (Figure 2). We consider a gene with a TPM of 3 or less to not be expressed in these cells. Importantly, our expression data is for wildtype HAP1 cells, knockout cell lines’. As gene expression profiles in a cell might change when a knockout is produced, RNA and protein expression profile data should be verified and characterized once the cell line is received.

In our characterization of genes in HAP1, we have also determined a set of genes that we deem highly essential and the genes that are diploid in these cell lines. Because of the essential nature of these genes or the presence of more than copy of this gene, their knockout cell lines will not appear for sale on our website. If you cannot find a listing for the gene of interest, contact scientific support to inquire further.

Additional characterization including of sequencing of HAP1 cells has been performed independently. (Essletzbichler et al 2014:)

How are HAP1 knockout cells produced? And how do you ensure a knockout has been obtained?

Our HAP1 knockout cells are produced using CRISPR/Cas9 technology. In this process, a guide RNA (gRNA) forms a complex with Cas9 nuclease, and directs Cas9 to a specific locus, where it cleaves the DNA. The cut is repaired by non-homologous end joining, often resulting in indels (small insertions and deletions). Indels can cause frameshifts, which usually create a premature stop codon in the gene. These cells are grown out clonally and then sequenced at the genomic DNA level to ensure a frameshift mutation is present. This disrupted sequence leads to a premature stop codon resulting in NMD of the transcript or a frameshift mutation resulting in a protein of a different composition). The gRNA used and the Sanger sequencing result can be found on the certificate of analysis (CoA) of the cell line. Both the gRNA and Cas9 are expressed transiently in the cells, so the cells can be best used as you see fit moving forward as these components will be lost from your final produced cell line. More about how these knockouts are produced can be found in this video.

Since every researcher’s assay is different, we recommend further characterization of the cell line upon receipt to see how the knockout cell is morphologically and functionally different to the wildtype.

Will the HAP1 cells diploidize?

HAP1 cells do spontaneously diploidize, and this is a normal part of their biology. It can normally be observed starting around passage 10, with some HAP1 cell lines completely becoming diploid by passage 20. We produce the knockout in the cells when they are haploid, so diploidization after this point will just result in the other allele having a copy of the knockout produced. The knockout status of the cell will not be affected by diploidization.

It is possible to isolate and propagate haploid HAP1s from the cell line, as most populations are a mixed pool of haploid and diploid cells. The haploid HAP1s are roughly half the size of the diploid HAP1s. If a haploid population is desired or needed for a particular assay it is recommended to use the HAP1 cells from passages from 7 to 10 and do an enrichment of HAP1s by size by flow cytometry. Freezing down cells at an early passage will also make it easy to return to this haploid population. More about the differences between the Haploid and diploid HAP1s can be read about in this paper Beigl et al. Biology Open. 2020

How can I differentially detect a knockout from the wildtype cell line?

CRISPR/Cas9 is used create our HAP1 knockouts, by introducing a premature stop codon or frameshift early in the gene. of this technique, some detection methods might fail to resolve the difference between the knockout and the parental cell line. The best way to validate your knock out is using a well-characterized functional assay that can delineate these differences between the mutant and parental cell line.

Detecting mRNA by RT-PCR might reveal the transcript with the indel without regard to function. Validating the cell line with an antibody can also be challenging if the precise epitope of the antibody is not known and characterized. (See this article about validating your antibody). For a complete description of how the protein can be affected after knockout please visit this video.

What are the culturing requirements for HAP1?

HAP1 cells have specific culture requirements and should be cultured according to provided protocols. HAP1 cells should remain at or below 75 percent confluency. The doubling time is approximately 12-16 hours depending on precise culturing conditions. Note that, although HAP1 cells are adherent, we have observed them to ball up and detach from the plate when they duplicate their DNA in S-phase. When thawing cells or isolating single clones, performing half-media changes to ensure these cells are left behind and allowed to continue to divide. The HAP1 culturing protocol can be found here. In addition we have another blog post that will “set yourself up for success with HAP1 cells” that goes over additional points on maintaining these cells.

How can I use a HAP1 cell line in my study?

HAP1s are a powerful research tool. HAP1 knockouts can be important for antibody validation, or they can be used as proof of concept before creating a more complex knockout. For some additional ideas how HAP1s have been used, check out this blog post “Beyond the Western Blot”

If you have further questions about the HAP1 knockout cells feel free to reach out to Scientific support by phone, chat, or at technical@horizondiscovery.com and we can discuss the potential to use HAP1s in your assay.

Learn more with these resources

• Why are there several HAP1 cell lines for my gene, how long will the take to be delivered Blog Article
• Good experiments with HAP1 cells: parental and mutant cell lines, media controls, assay validation, ploidy Blog article
• Beyond the western blot and the advantages of HAP1 cells Blog article
• HAP1 cell references reading list Reading list
• Top peer reviewed scientific articles using HAP1 cells Blog article
• HAP1 FAQs Blog article

References

1. Essletzbichler P. et al., Genome Res. 2014. Genomic characterization of HAP1 cell line.
2. Dong M. et al., Neurology 2014. HAP1 knockout cell line for evaluation of pathogenic mutations using phenotype rescue experiments;
3. Kravtsova-Ivantsiv Y. et al., Cell 2015. HAP1 knockouts of KPC1 and KPC2 support role of KPC1 as E3 ligase that mediates processing of NF-kB1 p105 to p50; Carette et al. Nature. 2011.
4. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1; Lackner DH et al. Nat Commun. 2015.
5. A generic strategy for CRISPR-Cas9-mediated gene tagging. Essletzbichler, Patrick et al. “Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line.” Genome research vol. 24,12 (2014): 2059-65.
6. Beigl et al. Efficient and crucial quality control of HAP1 cell ploidy status. Biology Open. 2020