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It’s a knock-out: KO cell lines in assay development

Related

  • Knock-out cell lines
    • Applying CRISPR to the drug discovery pipeline
      • Using CRISPR to speed the drug pipeline
        • KO validation to improve the R&D pipeline
          • How KO cell lines can be used
            • Five pillars to determine antibody specificity

              We talked to some of our expert cell lines team in order to understand how knock-out (KO) cell lines are streamling assay development in the drug discovery process. 

              Published February 24, 2021

              Bringing a new therapeutic all the way from a bright idea to market authorization is a long and difficult journey, with many points of failure along the way. 

              Most novel drugs never make it through clinical trials, often due to a lack of efficacy or unacceptable side effects, representing a waste of time, money, and, ultimately, the lives and health of the patients who need them.

              There have been significant efforts to de-risk this process by improving the validation of potential therapeutic targets and mechanisms of action, along with early identification of likely off-target effects and toxicity.

              At the heart of this challenge lies the need to develop and optimize assays that provide researchers with the data they require to identify potentially bioactive compounds and understand how they’re working - or why they’re not working - as efficiently and cost-effectively as possible. And to do that, you need the right tools.

              “So many candidates fail in clinical trials, or don’t even get there in the first place, because of problems confirming the connection between target and outcome,” says Aqsa Meer, Senior Product Manager at Abcam. 

              “Developing informative, robust, and biologically relevant assays that give you clear answers is a vital part of this process.”

              Contents

              • Take control of your assay development
              • You don't have do it yourself!
              • Say goodbye to cell hell 
              Bringing a new therapeutic all the way from a bright idea to market authorization is a long and difficult journey, with many points of failure along the way. 

              Most novel drugs never make it through clinical trials, often due to a lack of efficacy or unacceptable side effects, representing a waste of time, money, and, ultimately, the lives and health of the patients who need them.

              There have been significant efforts to de-risk this process by improving the validation of potential therapeutic targets and mechanisms of action, along with early identification of likely off-target effects and toxicity.

              At the heart of this challenge lies the need to develop and optimize assays that provide researchers with the data they require to identify potentially bioactive compounds and understand how they’re working - or why they’re not working - as efficiently and cost-effectively as possible. And to do that, you need the right tools.

              “So many candidates fail in clinical trials, or don’t even get there in the first place, because of problems confirming the connection between target and outcome,” says Aqsa Meer, Senior Product Manager at Abcam. 

              “Developing informative, robust, and biologically relevant assays that give you clear answers is a vital part of this process.”


              Take control of your assay development

              Assays have a part to play in many phases of the drug development journey, from high-throughput functional or phenotypic screens designed to detect promising hits through to validating targets and binding sites, uncovering the mechanism of action, and investigating toxicity. 

              “When you first start on the drug discovery pathway, you can get up to 15 or 20 potential hits, so you want to know which ones of them are real before you go any further,” explains Julie Wickenden, Head of Cell Sciences at Abcam. 

              “Then you start digging into exactly how your lead compounds are working. Which target or targets are they acting on, and does that have the biological activity you’re looking for? Is the binding highly specific, or are there off-target effects? Whatever your assay, you need to be sure you’re working with the right reagents if you want to get reliable, meaningful data.”

              Whether an assay is based on flow cytometry, microscopy, immunohistochemistry, HPLC, or any other technique, it’s essential to have a suitable negative control. This could be in the form of a cell type that is known not to express the putative target or cells that have been treated in some way to reduce or remove activity.

              This reduction can be achieved through chemical inhibitors or RNA knockdown to reduce gene expression, although both approaches can leave residual activity. A better option is to use a knock-out cell line, where the gene of interest is gone completely. 

              “One of the biggest challenges that assay developers face is getting the right controls, whether that’s positive or negative,” says Karine Enesa, Head of Cell Line Development at Abcam. 

              “Using knock-out cells in assays is a true negative control that helps you get a much cleaner signal with less noise than using an inhibitor or knockdown - you can’t get a better negative control than a cell line with no protein at all.”

              Don’t do it yourself

              The advent of CRISPR and other gene-editing technologies has made it easier than ever to make knock-out cell lines for drug development assays, but unless you’re lucky enough to work somewhere with a dedicated high-throughput knock-out facility, it can take many months and a lot of frustration to get the line you want.

              “Making your own knock-out cell line may look simple on paper, but planning, designing, and testing different CRISPR guides, followed by validation of your lines, can be tricky, time-consuming, and expensive,” says Julie. 

              “That quickly adds up if you need multiple different knock-outs or cell backgrounds for your experiments, so it’s much quicker and cheaper to buy in the lines you need rather than make them from scratch.” 
                
              Abcam’s catalog of nearly 3,000 ‘off the shelf’ knock-out cell lines opens up exciting opportunities for assay developers. For example, there are knock-outs covering every component of several biologically relevant pathways, such as MAP kinase or apoptotic pathways, to help drug developers home in on their target and mechanism of action. 

              As well as being the gold standard for antibody validation, knock-out lines can also reveal unexpected off-target drug or antibody binding, which may lead to unanticipated toxicities or side effects further down the road. 

              Knock-out lines can also be used for synthetic lethality screens, rescue assays, co-culture experiments, or as a convenient base for making further double or triple knock-outs. 

              Say goodbye to cell hell

              Abcam’s extensive and ever-growing knock-out cell line and cell lysate catalog covers a wide range of immortalized mammalian cell types and targets for research use only, with a focus on oncology, immuno-oncology, and neuroscience.
              The team have also responded rapidly to the needs of the research community by producing a range of cell lines for COVID-19 research, including ACE2 knock-outs.
              Every line is certified mycoplasma-free and has good viability, with advice on culturing tips and techniques available on hand from Abcam’s experts. All knock-outs are validated with Sanger sequencing, and many are also validated at the protein level. Importantly, every knock-out also comes with the matching background wild type cell line for reliable comparisons.
              If the target or cell type you’re looking for isn’t already available, Abcam offers a custom service for any background of your choice.
              “If you don’t see it in our catalog, just ask for it,” says Karine. “Our expert team in Fremont, California, are able to do custom knock-outs, knock-ins, point mutations, overexpression, underexpression, double knock-outs, and more. The timelines vary, depending on the complexity of the project, but it’s still much more efficient than doing it yourself.”

              Whether it’s custom or off-the-shelf, having easy access to a wide range of knock-out cell lines can significantly accelerate and streamline the process of developing and optimizing assays for drug development. 
              Robust assays that can generate reliable, informative data ultimately increase the chances of later success in clinical trials and getting to market faster with life-changing therapies for patients.
              “One researcher was in the middle of making his own knock-out cell line, but he abandoned it immediately when he realized he could just buy ours,” Aqsa says. 
              “If you ordered a cell line today, you could be doing experiments in a fortnight. Why would you spend all your time making reagents when you could move straight to doing assays?”

              Take control of your assay development

              Assays have a part to play in many phases of the drug development journey, from high-throughput functional or phenotypic screens designed to detect promising hits through to validating targets and binding sites, uncovering the mechanism of action, and investigating toxicity. 

              “When you first start on the drug discovery pathway, you can get up to 15 or 20 potential hits, so you want to know which ones of them are real before you go any further,” explains Julie Wickenden, Head of Cell Sciences at Abcam. 

              “Then you start digging into exactly how your lead compounds are working. Which target or targets are they acting on, and do they have the biological activity you’re looking for? Is the binding highly specific, or are there off-target effects? Whatever your assay, you need to be sure you’re working with the right reagents if you want to get reliable, meaningful data.”

              Whether an assay is based on flow cytometry, microscopy, immunohistochemistry, HPLC, or any other technique, it’s essential to have a suitable negative control. This could be in the form of a cell type that is known not to express the putative target or cells that have been treated in some way to reduce or remove activity.

              This reduction can be achieved through chemical inhibitors or RNA knock-down to reduce gene expression, although both approaches can leave residual activity. A better option is to use a knock-out cell line, where the gene of interest is gone completely. 

              “One of the biggest challenges that assay developers face is getting the right controls, whether that’s positive or negative,” says Karine Enesa, Head of Cell Line Development at Abcam. 

              “Using knock-out cells in assays is a true negative control that helps you get a much cleaner signal with less noise than using an inhibitor or knock-down - you can’t get a better negative control than a cell line with no protein at all.”

              You don’t  have to do it yourself!

              The advent of CRISPR and other gene-editing technologies has made it easier than ever to make knock-out cell lines for drug development assays, but unless you’re lucky enough to work somewhere with a dedicated high-throughput knock-out facility, it can take many months and a lot of frustration to get the line you want.

              “Making your own knock-out cell line may look simple on paper, but planning, designing, and testing different CRISPR guides, followed by validation of your lines, can be tricky, time-consuming, and expensive,” says Julie. 

              “That quickly adds up if you need multiple different knock-outs or cell backgrounds for your experiments, so it’s much quicker and cheaper to buy in the lines you need rather than make them from scratch.” 

              Our catalog of nearly 3,000 ‘off the shelf’ knock-out cell lines opens up exciting opportunities for assay developers. For example, there are knock-outs covering every component of several biologically relevant pathways, such as MAP kinase or apoptotic pathways, to help drug developers home in on their target and mechanism of action. 

              As well as being the gold standard for antibody validation, knock-out lines can also reveal unexpected off-target drug or antibody binding, which may lead to unanticipated toxicities or side effects further down the road. 

              Knock-out lines can also be used for synthetic lethality screens, rescue assays, co-culture experiments, or as a convenient base for making further double or triple knock-outs. 

              Say goodbye to cell hell

              Our extensive and ever-growing knock-out cell line and cell lysate catalog covers a wide range of immortalized mammalian cell types and targets, with a focus on oncology, immuno-oncology, and neuroscience.

              Our team have also responded rapidly to the needs of the research community by producing a range of cell lines for COVID-19 research, including ACE2 knock-outs.

              Every line is certified mycoplasma-free and has good viability, with advice on culturing tips and techniques available on hand from our experts. All knock-outs are validated with Sanger sequencing, and many are also validated at the protein level. Importantly, every knock-out also comes with the matching background wild type cell line for reliable comparisons.

              If the target or cell type you’re looking for isn’t already available, we also offer a custom service for any background of your choice.

              “If you don’t see it in our catalog, just ask for it,” says Karine. “Our expert team in Fremont, California, are able to do custom knock-outs, knock-ins, point mutations, overexpression, underexpression, double knock-outs, and more. The timelines vary, depending on the complexity of the project, but it’s still much more efficient than doing it yourself.”

              Whether it’s custom or off-the-shelf, having easy access to a wide range of knock-out cell lines can significantly accelerate and streamline the process of developing and optimizing assays for drug development. 

              Robust assays that can generate reliable, informative data ultimately increase the chances of later success in clinical trials and getting to market faster with life-changing therapies for patients.

              “One researcher was in the middle of making his own knock-out cell line, but he abandoned it immediately when he realized he could just buy ours,” Aqsa says. 

              “If you ordered a cell line today, you could be doing experiments in a fortnight. Why would you spend all your time making reagents when you could move straight to doing assays?”


              Interested in learning more about our knock-out cell lines? 

              Explore our ready-to-use knock-out cell lines or custom gene-editing services.







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