HAP1 Cell Line Applications

The following scientific publications showcase how gene edited HAP1 cell lines are being used in research. These highlight the wide range of applications from genetic screens through to de-convolution of findings from patient derived samples.

Tyrosine kinase transcriptome‐based reverse genetic screen: Collection of knockout HAP1 cell lines for systematic multiplexed phenotyping.

This study used a scalable reverse genetic approach based on multiplexed RNA sequencing of gene edited HAP1 cells, with knockouts covering tyrosine kinases. The authors conducted 10 parallel screens and identify known and unexpected effects on signaling pathways. This proof of concept demonstrates the application of collections of knockout HAP1 cells lines in the systematic phenotyping of human genes and uncharacterized genomic features linked with human disease.

Reference Mol Syst Biol. (2016) 12: 879, Gapp et al.  Parallel reverse genetic screening in knock out HAP1 cells lines using transcriptomics


NF-κB1 p105 processing to tumor-suppressing p50 is mediated by KPC1 Ubiquitin ligase.

Limited proteosomal processing of NF-kB1 precursor p105 produces the active p50 subunit, a transcriptional activator of tumor suppressive genes, hence leading to inhibition of tumor growth. The process is mediated by the ubiquitin (Ub) system, where KPC1 was recently identified as the essential Ub ligase using chromatography and cell-free conjugation assays.

Reference: Cell. 2015 Apr 9;161(2):333-47. Kravtsova-Ivantsiv et al KPC1-mediated ubiquitination and proteasomal processing of NF-κB1 p105 to p50 restricts tumor growth


Docetaxel sensitivity is enhanced by loss of SW1/SNF chromatin remodeler component SMARCA4.

Chromatin regulators are known to affect tumorogenesis, tumor hetergenity, and cellular responses to cancer therapies (radio- and chemotherapy). A functional association between the SW1/SNF chromatin remodeler and the docetaxal response was extrapolated from gene expression data, and observed in NC-60 cell lines. A key partner of SW1/SNF is SMARCA4. 

Knocking out SMARCA4 in HAP1 cells resulted in a 4-fold decrease in cell survival when exposed to docetaxal. Thus SW1/SNF could be a potential biomarker for docetaxal sensitivity, as well as a therapeutic target, perhaps by interfering with partners such as SMARCA4

Reference:  Mol Cancer Therapy 2016. Gurard-Levin et al. Chromatin regulators as a guide for cancer treatment choice 


TSSC1 is identified as a novel component of the endosomal retrieval machinery, using HAP1 wildtype, knockout, and rescued knockout cell lines.

Wildtype, TSSC1 KO and TSSC1 KO rescued HAP1 cells were used at several points in this body of work, alongside H4, Hela and rat brain cells. HAP1s were specifically used to validate an antibody and to test the involvement of TSSC1 in retrograde transport from endosomes to the trans-Golgi network.  

Reference: Mol Biol Cell. 2016  Gershlick et al. TSSC1 is Novel Component of the Endosomal Retrieval Machinery


p53 in α-synuclein gene activation in Parkinson’s disease.

Accumulation of α-synuclein in Lewy bodies plays an important role in Parkinson’s disease. Pharmacological challenge and transient overexpression of p53 in the dopaminergic neuroblastoma cell line SH-SY5Y indicated that p53 increases α-synuclein mRNA and protein levels. A knockout p53 HAP1 cell line confirmed the role of p53 in elevating α-synuclein gene expression, suggesting potential new approaches to designing neuroprotective strategies in Parkinson’s disease.

Reference: Molecular Neurodegeneration (2016) 11:13. Duplan et al. Direct α-synuclein promoter transactivation by the tumor suppressor p53.  


CASD1 is a key sialic acid 9-O-acyltransferase

Modification of sialic acids on glycoproteins and lipids plays an important role in development, cellular recognition and host-pathogen interactions; their 9-O-acetylation has been implicated in cell survival in acute lymphoblastic leukemia (ALL). 

In HAP1 cells, gene knockout of CASD1 completely abolished 9-O-acetylation of sialoglycans in the Golgi. Similarly, the crucial role played by CASD1 in 9-O-acetylation of cell surface gangliosides was demonstrated in these HAP1 cells. Not only providing insights into the catalytic mechanism of 9-O-acetylation, this highlights CASD1 as a potential target in therapies to combat drug resistance in ALL.

Reference: Baumann et al. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate.


POMGNT2 mutations are involved in the pathogenicity of milder forms of muscular dystrophy.

In patients with dystroglycanopathy (DGP) two missense variants of POMGNT2 were identified using whole-exome sequencing. Transient expression of wt or the two mutant cDNAs in HeLa and HEK 293 cells determined POMNT2 protein localization and activity. Knockout POMGNT2 HAP1 cells transfected with wt cDNA or wt HAP1 cell controls retained the ability to hypoglycosylate α-dystroglycan, as detected by recognition by the anti-α-dystroglycan antibody, IlH6. In contrast, the mutated POMNT2 cDNAs transfected into knockout POMGNT2 HAP1 cells failed to rescue the defective ILH6 recognition phenotype. This method allows researchers to verify the pathogenesis of variants in known or novel candidate genes associated with DGP without using patient material.

 Reference: Neurol Genet 2015. Endo et al. Milder forms of muscular dystrophy associated with POMGNT2 mutations.


DAG1 involvement in milder forms of muscular dystrophy confirmed with genetic rescue.

Whole-exome sequencing revealed two missense mutations in the dystroglycan-encoding gene DAG1 in one patient. The pathogenicity of each mutant was evaluated directly in gene-modified HAP1 cells because patient cells were not available. Knockout DAG1 HAP1 cells failed to be recognized by the anti-α-dystroglycan antibody, IlH6. Only the wt, but not the mutant DAG1 cDNAs rescued ILH6 immunoreactivity back to levels seen in wt HAP1 cells. However, the DAG1 mutants did not affect β-dystroglycan transport to the cell surface in DAG1 KO cells, as seen by in situ immunostaining and cell surface biotin labelling. Apparently these mutations impair β-dystroglycan glycosylation, but not dystroglycan expression, processing and transport. Such phenotypic rescue experiments provide a powerful means to evaluate pathogenic mutations in the broad spectrum of dystroglycanopathies.

Reference:  Neurology 2015. Dong M. et al. DAG1 mutations associated with asymptomatic hyperCKemia and hypoglycosylation of α-dystroglycan 

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