1. What type of cells are KBM-7 cells?
KBM-7 is a cell line isolated from a patient with Chronic Myelogenous Leukemia (see Kotecki et al. Experimental Cell Research 1999). These cells are similar to myeloid progenitor cells and grow in suspension. They have a BCR-ABL fusion oncogene and a mutation in p53.
2. How do you culture KBM-7 cells?
KBM-7 cells are suspension cells cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) in the presence of 10% fetal calf serum and penicillin/ streptomycin. Cells are typically seeded at a density of 2x106 cells/ml and grow with a doubling time of roughly 24h. Cells should be passaged every 2-3 days.
3. How do you introduce mutations in KBM-7 cells?
We employ a gene trapping strategy that introduces retroviral insertional mutations across the genome of KBM-7 cells. The principal elements consist of a 3’ splice site (splice acceptor SA), a downstream transcriptional termination sequence (polyadenylation sequence, poly A) and a promoter-less enhanced GFP open reading frame. Integration of the retroviral gene trap into a gene can lead to (i) insertion into an exon which disrupts the coding sequence and introduces a premature stop codon, (ii) insertion into an intron which disrupts splicing, or (iii) ) insertion into an intron which leads to premature transcriptional termination.
The gene trap contains molecular barcodes and loxP sites. A detailed overview as well as the sequence of the gene trap after integration into the host genome can be downloaded here
- Map of gene trap after integration into the human genome (Download map)
- Gene trap sequence after integration into the human genome (Download sequence)
The Human Gene Trap Mutant Collection was built using several different loxP site configurations illustrated here (Download illustration).
4. Is my favorite gene expressed in KBM-7 cells?
KBM-7 cells have been characterized by Whole Genome Sequencing (DNA) and RNA sequencing. For any given gene, Haplogen reports an FPKM (“fragments per kilobase of exon per million fragments mapped”) value, obtained from KBM-7 wild type cells. Genes with FPKM values of >3 are considered to be expressed, while genes with FPKM values between 0.05 and 3 are considered to be expressed at low levels. Genes with values below 0.05 are considered not to be expressed in KBM-7 cells. To allow easy and up-to-date access to the above described data sets, we created a UCSC Genome Browser hub that can be accessed here:http://kbm7.genomebrowser.cemm.at/
5. How do you map the genomic integration site of the gene trap?
We use sequencing reads of 50-70 bases derived from inverse PCRs. These reads are mapped to the human genome and when they align uniquely to a human gene, the corresponding clone qualifies as a mutant of that particular gene. As our clones are mapped in a high throughput procedure, we recommend that you also verify our alignments manually.
6. I want to use a mutant clone. Which controls do you recommend?
We send out “reference clones” with every gene-trap order that can be used as a control in your experiments. Reference clones have a history identical to our mutant clones including retroviral transduction, but bear insertions in intergenic regions on (diploid) chromosome 8 and are thus highly unlikely to affect gene function. In addition, we recommend reconstitution of the mutant clone with the respective cDNA. This should revert the phenotype of the mutant clone. Finally, you may want to exploit the loxP sites that allow excision or inversion of the gene trap cassette (see FAQ 12 for more detailed information).
7.How should I validate whether the gene trap disrupts gene expression?
The impact of the gene trap on the expression of a gene is best assessed at the protein level, e.g. by Western Blotting. This is sometimes difficult and depends on the availability of antibodies. As a general validation we recommend quantitative RT-PCR (qRT-PCR) as a surrogate model.
Caution! The outcome of qRT-PCR is dependent on the primer design!
We obtain the most reliable results when using primers that flank the gene trap insertion site (ie. one primer upstream and one primer downstream of the gene trap insertion).
It is also possible to use primers that bind downstream of the gene trap insertion, but we observed that the data obtained with downstream primers are less predictive than those obtained with flanking primers.
As example, we provide data on the clone 25B07 (affecting BTK; download here).
8. How are mutant clones validated?
Customer satisfaction is our top priority. Therefore, all our clones are subjected to rigorous quality control.
Mutant clones are validated by quantitative RT-PCR.
Inducible clones are clones bearing a gene trap insertion in the inverse orientation. Upon addition of Cre (by the customer), the gene trap is inverted and the corresponding gene is disrupted. For inducible clones, we verify the genomic integration site of the gene trap cassette.
9. How do you make sure a given “clone” is really derived from a single cell clone?
Clonality of our cells can be assessed based on the molecular barcode: every clone harbors a unique DNA barcode that can be amplified by PCR and read by Sanger sequencing (see Protocol “Amplification and sequencing of molecular barcodes” for more details). If a clone was contaminated with another clone, the Sanger sequencing would not report one single barcode, but show ambiguities in the barcode region. If that happens, please report back to us. You may decide to subclone that particular “clone” to obtain a clonal population of cells.
10. Can you be sure that a given clone only bears one gene trap insertion (as opposed to several)?
Yes. We generate our clones by retroviral transduction of KBM-7 cells. To prevent multiple gene trap insertions per cell, we use a low dose of retrovirus. In addition, our clone mapping procedure assesses whether any given clone bears one or multiple insertions. Clones with multiple insertions are discarded.
11. KBM-7 cells bear molecular barcodes. What can these be used for?
Each of our clones is marked by a unique 22 bp DNA barcode. This barcode can be used to confirm the identity as well as the clonality of any given clone (see also FAQ 9). In addition, the barcode can be used to track the fitness of any given clone in a mixture of clones.
12. What can the loxP sites be used for?
Most of our clones were generated using a gene trap vector with loxP sites. These can be used to remove the gene trap and hence revert the phenotype of the mutant. Several gene trap versions were used with different loxP site configurations:
- Parallel loxP sites: enable Cre-mediated excision of the gene trap.
- Antiparallel loxP sites: enable Cre-mediated inversion of the gene trap (multiple rounds of inversion are possible).
- Paired incompatible loxP/lox511 sites: enable Cre-mediated unidirectional inversion of the gene trap (only a single inversion possible).
An illustration of the different loxP site configurations can be downloaded here
13. Are there any restrictions for publishing results obtained with mutant clones?
Results obtained with mutant clones can be used for publication without restriction. We kindly ask you to acknowledge the source of the clones appropriately, by stating that “Mutant clones used in this study were purchased from Horizon Discovery (Cambridge, UK) and generated by Haplogen (Vienna, Austria)” and by citing the following reference:
Bürckstümmer T, Banning C, Hainzl P, Schobesberger R, Kerzendorfer C, Pauler FM, Chen D, Them N, Schischlik F, Rebsamen M, Smida M, de la Cruz FF, Lapao A, Liszt M, Eizinger B, Guenzl PM, Blomen VA, Konopka T, Gapp B, Parapatics K, Maier B, Stöckl J, Fischl W, Salic S, Taba R, Knapp S, Bennett KL, Bock C, Colinge J, Kralovics R, Ammerer G, Casari G, Brummelkamp TR, Superti-Furga G and Nijman SMB. A reversible gene trap collection empowers haploid genetics in human cells. Nature Methods 10, 965–971 (2013) doi:10.1038/nmeth.2609
14. My shipment came thawed! How do I get a new delivery?
Please send an email to firstname.lastname@example.org and request a new shipment.
15. Can the results be generalized to other model systems?
After using this approach for over three years, we have not encountered a single phenotype for which data obtained in KBM-7 did not translate into other cellular model systems. Please find publications which have successfully used haploid cells for genetic studies in the literature section.
16. Are KBM-7 cells fully haploid?
KBM-7 are haploid for all human chromosomes, except chromosome 8 and parts of chromosome 15. We therefore refer to KBM-7 as “near-haploid” cells.
17. How do you determine whether or not a given batch of KBM-7 cells is near-haploid?
The ploidy status is typically assessed by propidium iodide staining coupled to flow cytometry. Overnight incubation with colcemide (100 ng/ml final concentration), which prevents mitotic cell division, may be used to enhance the 2N peak. KBM-7 cells should be compared to a well-established diploid cell line, such as HCT116 (ATCC, CCL-247). Please note that even if your clone has reverted to a diploid genome, it will still be a homozygous mutant.
18. How stable is the near-haploid genotype of KBM-7 cells?
The near-haploid genotype of KBM-7 cells is remarkably stable: KBM-7 cells should stay near-haploid for weeks if continuously passaged (Kotecki et al. Experimental Cell Research 1999). When diploid cells arise, the population can be subcloned to obtain near-haploid cells. Alternatively, near-haploid cells can be isolated using flow cytometry by sorting for small cell size.