Revealing the role of E3 ubiquitin ligases in DNA damage repair

One of the diverse new uses for the HAP1 cell line, one that has begun to draw significant attention, is in the field of DNA damage repair. A recent paper from Minoru Takata’s group highlights this important application of this relatively new tool.

The dilemma of when to invest in new technology

Researchers in the life sciences community are constantly walking a fine line in assay development. On one side is the accuracy, specificity and reproducibility borne from use of a well-established tool; i.e., a tool that has been on the market for a long time. Put another way, there is a level of comfort in using the same products for many years - in science as in the rest of life.

On the other side is the importance of finding the most efficient, cost-effective methods to carry out experiments. Doing so often means taking a chance on a new product, running it alongside existing methods to compare. Of course, it’s not just cost-effectiveness that necessitates making changes; simply keeping up can mean bringing in a new product that incorporates new advances. The outcome, hopefully, is better results faster, at lower cost.

And yet, inertia is a challenging force to overcome, and there is always a tendency to maintain the status quo. Particularly, as noted above, when so much rides on maintaining consistent protocols.

Here at Horizon, our scientists have built a remarkable new tool in the HAP1 cell line to facilitate researcher's access to CRISPR technology. These knockout cell lines allow researchers to quickly validate their gene or target of interest, without having to invest time and resource in developing in-house CRISPR technology.

HAP1 and HAP1 cells gene-edited to knockout SLC30A6 (HAP1_SLC30A6, catalogue number: HZGHC002784c010) with the HPA antibody HPA057328 targeting SLC30A6 demonstarting the specificity of this antibody. The samples were prepared in parallel using the same antibody dilutions and reagents, and both images are acquired with the exact same settings. Images curtesy of Dr Emma Lundberg, Cell Profiling facility. KTH Royal Institute of Technology.

We believe that, for its designated applications, HAP1 cells are more than worth adding into a lab’s toolbox. However, our opinion only takes things so far. So we set out to ask a few scientists who have published using the HAP1 cells about their work, and how the cells played a role in their investigations.

Read our blog on how new methods increase precision in protein visualization

Here we describe some of the great solutions that are coming out of recent advances using CRISPR CAS technology that give more precise and physiological results for protein visualization.  In our previous blog (see link at end of article), we discussed the some of the difficulties  with traditional methods for protein tracking and localization. One of the main causes of variability and wasted resources is the lack of standards for antibody quality. 

To be useful, an antibody must:

• Be specific

• Have a high signal to noise ratio

• Be validated for the assay at hand

Efforts to reduced non-specific antibodies in both industry and academia

From an industry standpoint, numerous organizations and commercial suppliers have created (or are creating) programs to ensure that the above criteria are met for each new antibody brought to market.

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 selected articles show the broad applicability of the HAP1 cell line, and provide characterization data to help your research. If you would like to know more, please follow these links to our ready made cell lines and cell line engineering services.

Publications:

2017

• Measurement of nanoscale DNA translocation by uracil DNA glycosylase in human cells
• CRISPR/Cas9-Mediated Scanning for Regulatory Elements Required for HPRT1 Expression via Thousands of Large, Programmed Genomic Deletions
• Trap^Seq: An RNA Sequencing-based pipeline for the identification of genetrap insertions in mammalian cells
• Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia
• RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination
• eIF1 modulates the recognition of suboptimal translation initiation sites and steers gene expression via uORFs
• Nit1 is a metabolite repair enzyme that hydrolyzes deaminated glutathione
• FBXO25 regulates MAPKsignaling pathway through inhibition of ERK1/2 phosphorylation
• Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase
• Translation of Sindbis Subgenomic mRNA is Independent of eIF2, eIF2A and eIF2DJ
• Stress-specific differences in assembly and composition of stress granules and related foci

2016

• Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling
• Recycling of Apolipoprotein(a) After PlgRKT-Mediated Endocytosis of Lipoprotein(a)
• The RNA-binding protein Secisbp2 differentially modulates UGA codon reassignment and RNA decay
• The Selenocysteine-Specific Elongation Factor Contains Unique Sequences That Are Required for Both Nuclear Export and Selenocysteine Incorporation
• Dual loss of succinate dehydrogenase (SDH) and complex I activity is necessary to recapitulate the metabolic phenotype of SDH mutant tumors
• Parallel reverse genetic screening in mutant human cells using transcriptomics

Sensitivity and resistance to DNA Damage Response Pathways identified with gene-edited cell lines and wildtype controls

The cellular DNA damage response (DDR) is an essential safeguard against cancer. Upon activation, the DDR can limit tumour progression at the early stages by inducing senescence or cell death. When this defence fails tumors are able to develop. However, with time, tumors accumulate more mutations in DNA repair proteins as cancers progress.  The efficiency of DDR plays an essential role in the effectivity of cytotoxic treatments. Currently much research is focussed on identifying the DDR mechanisms involved in cancers and how these dysfunctional processes can be utilized against tumor growth.

When I have read articles just like this one early on in my career, I would laugh and categorize it with blogs regarding Bigfoot and the Loch Ness Monster. However, much has changed in the past 10 years. New technologies have been developed and milestones have been reached that should have Cancer a little worried. These 3 steps might be viewed to some as obvious, but I argue that it’s how the researchers have utilized the technology wisely that has made the difference. I have identified some papers that have carved a successful path to Cancer's possible demise.

One of the highest ideals in science is to observe natural events in their native context. Doing so is a constant challenge thanks to the Observer Effect, described by Heisenberg and others, where the act of observing or measuring a process alters it. Thus, scientists of all stripes try to get out of the way, attempting to produce the most accurate measurements possible using specific yet unobtrusive tools. Wildlife photographers use long-range lenses to avoid the need to stand directly in front of a herd of water buffalo and thereby affect the animals’ behaviour. Psychologists create tests where the subjects are unaware of the true intent so as to minimize changes in natural responses.

Knockout and tagged gene-editing can create cell lines ideal for antibody validation. Using CRISPR CAS technology, Horizon developed a streamlined process to cut production time. Read more in our interview with Lead Scientist Daniel Lackner

Audio also available

The visualisation of proteins or organelles in cells and other complex biological systems is ‘bread and butter’ stuff for cellular and molecular biologists; it’s performed day-in, day-out in labs across the globe. But this doesn’t mean that the most popular approaches currently used for protein visualisation – dye staining, antibody labelling and fusion protein over-expression systems – are ideal. Almost every scientific technique has its advantages and drawbacks, and the various options currently available for protein visualisation are not exceptions to this rule, so let’s have a look at the pros and cons of each.

Recycling has always been a smart idea, and nature has its own processes to ensure that waste is kept to a minimum.  As Professor Ohsumi discovered, autophagy is the cells way of degrading and recycling cellular components, allowing it to adapt to nutritional deficiency or other environmental influences. Professor Yoshinori Ohsumi, honorary professor and leader of the Cell Biology Unit at the Tokyo Institute of Technology, has been studying autophagy for 27 years. This year's Nobel Laureate discovered and elucidated mechanisms underlying autophagy, according to the Press Release from The Nobel Assembly at Karolinska Institutet.