Mammalian cell-based libraries offer a physiologically relevant context for high throughput screening of high affinity antibodies. Current technologies using retroviruses to develop mammalian cell libraries result in random, multiple copy integration at various loci of the genome. As a result, the expression level amongst different cells in the library is varied leading to false representation of the binding affinity of individual molecules. In this talk, we introduce the TARGATT™ site-specific, integrase-mediated gene integration technology which provides an efficient strategy for integrating a gene of interest into a preselected, transcriptionally active locus. A TARGATT™-HEK293 master cell line was generated to enable knock-in of only a single copy of the transgene in every cell at the same locus, with efficiency over 90%. The TARGATT™ system thus provides an efficient platform with uniform transgene expression and reproducibility for constructing large isogenic cell libraries for rapid, high throughput antibody screening. The same system can also be used for bioproduction, protein evolution, target validation and safety assessment in immuno-oncology, GPCR and ion channels.
How-to Guide for TARGATT™ Transgenic Kit, by Dr Ruby Chen-Tsai (December 2017)
The TARGATT™ Transgenic Kit is designed to create site-specific knock-in transgenic mice at a defined chromosomal locus in a more efficient and significantly faster way over traditional methods. Generating transgenic mice by conventional methods (e.g. pronuclear microinjection or lentiviral injection) has following limitations, first of which is random insertion of the transgene. Random insertion of a transgene results in position effect where either the transgene is prone to silencing or endogenous gene expression is disrupted. Secondly, transgenes can be inserted as multiple copies, resulting in instability at the insertion locus. Using our proprietary site-specific DNA integration system, TARGATT™, combined with our genetically engineered TARGATT™ mice (strain code 549), you can generate your desired trangenic mouse models with guaranteed gene expression in as little as three months.
This video gives a step-by-step overview on how to use the TARGATT™ Trangenic Kit and the TARGATT™ technology to generate site-specific knock-in mouse models.
Engineering of Isogenic Cell Lines Using the CRISPR/Cas9 Technology, and Precise Characterization of Low Allelic Frequency FFPE Cell Line Blocks for Use as Molecular Reference Standards (November 2017)
Cancer is a heterogeneous disease in which hundreds of genes, and many thousands of mutations, have been implicated in oncogenesis. As we enter an era of precision tumor profiling, there is a significant need for molecular reference standards that can be employed for assay development and quality assurance in order to validate assay performance, and understand cross-site and inter-operator reproducibility. Cell-line based reference standards are ideal for this application, since they represent a biologically-relevant, reproducible, and renewable source of control materials. With this in mind, we have engineered a reference bank of cell lines that contain over 250 oncogenic point mutations, insertions, and deletions, which are spread across multiple genes, including EGFR, KRAS, BRAF, PIK3CA, NRAS, HRAS, and TP53. Here, we present data on the utility of these cell lines for reference standard generation, using an engineered, EGFR C797S cell line as a case study. This mutation is an emerging resistance marker to EGFR T790M-targeted therapeutics.
Human induced pluripotent stem cell (iPSC) technology offers the benefits of a cell line coupled with the advantage of using human primary cells. Additionally, iPSCs are also amenable to genome editing, and engineered iPSCs and their isogenic control lines can be terminally differentiated into cells of multiple lineages. This presents an almost limitless access to relevant and predictive disease models for basic research, drug discovery, toxicity screening and hopefully for regenerative cell therapy. In this webinar, we will elaborate on a panel of iPSC lines engineered for neurotoxicity assays and disease modeling. The cell lines in this panel include: 1) control lines, 2) patient-specific lines, 3) lineage-specific knock-in reporters, 4) isogenic controls of single and double knock-outs. We have also established scalable protocols for generating differentiated cells in an assay ready format. This talk will focus on the utility of these lines for neurotoxicity assays, including assays to determine the specificity of different neural cell types for a small range of chemicals and drugs from the Tox21 library, as well as for neuroprotective assays with dopaminergic neurons.
Highlights of this webinar:
- Overview of iPSC genome editing and differentiation technology
- Panel of engineered iPSCs for neurotoxicity assays
- Neural differentiation and neurotoxicity/ neuroprotection screening assays
Choosing the Right Genome Editing Technology for Your Mouse Models: CRISPR, TARGATT™ and Beyond... (February 2017)
Highlights of this Webinar:
- Overview on available genome editing technologies in generating mouse models.
- Pros and cons of each technology
- Examples of animal and cell models generated by CRISPR and TARGATT™
- Next generation genome editing tools?
Autobioluminescent cells use a genetically encoded synthetic luciferase cassette to continuously produce a bioluminescent signal without the need for extracellular stimulation. By encoding both a luciferase protein, as well as a short synthetic pathway capable of transforming natural intracellular products into luciferin substrates, these cells can self-modulate their bioluminescent production in response to metabolic activity levels, or autonomously enact their bioluminescent phenotype in response to intra- or extracellular events. The use of this self-directed approach to bioluminescent imaging improves upon traditional reporters such as firefly luciferase (luc) by negating the need for light activating chemical substrate addition, which reduces the cost of performance while simultaneously increasing the amount of data that can be obtained per run. This eliminates the need for sample destruction or any investigator interaction, allowing for ultra-simplistic, low-cost bioluminescent screening using existing optical imaging equipment. This webinar will discuss the capabilities and uses of autobioluminescent cells for improving existing bioluminescent imaging workflows and for developing new workflows that leverage the autonomous signal generation phenotype to gather data not available from traditional optical imaging reporter platforms.
Highlights of this webinar:
- An introduction to autobioluminescence
- Autobioluminescent vs. bioluminescent imaging
- Using autobioluminescence for in vitro applications
- Using autobioluminescence for in vivo applications
- Autobioluminescent expression in stem cells
ONCOREF™ Reference Standards: Application of CRISPR/Cas9 to the Generation of Isogenic Cell Lines and Reference Materials (October 2016)
CRISPR/Cas9 is rapidly enabling the development of new tools for enhancing our understanding oncogenic mutations in cancer. In order to aid in advancing cancer diagnosis and treatment, Applied StemCell has recently engineered a series of 40 isogenic cell lines that feature diverse mutations in the MAPK pathway. These mutant lines are available as isogenic pairs for applications in lead compound discovery, or as FFPE and nucleic acid reference materials for assay development. This webinar will focus on ASC’s efforts in developing these research tools, as well as applications of the materials for the advancement of cancer research.
Highlights of this talk:
- Overview of molecular reference materials
- Workflow and QC for ONCOREF™ cell line generation
- Advantages of CRISPR-engineered molecular reference standards
- Applications of reference materials in assay development
- Q & A
This recorded webinar discusses Applied StemCell's efforts in fine tuning its CRISPR/Cas9 technology to improve the efficiency of gene editing in blood derived
Highlights of the talk: immune cells, such as Jurkat, K562, TF1, natural killer cell-derived cell lines and bone marrow cell lines. The talk also illustrates the potential difficulties when working with blood cells and demonstrates how Applied StemCell addresses these issues.
- Discrepancies in guide RNA (gRNA) activity among major blood-derived cell lines and how it affects gene editing efficiency
- Guidelines to generate your desired mutations when available gRNAs are not ideal
- Key steps to enhance your screening process and improve genome modification efficiency
- Alternate solutions for engineering cell lines sensitive to DNA plasmid transfection
This webinar introduces the TARGATT™ integrase based technology for generation of transgenic rats. The TARGATT™ platform enables very efficient insertion of large fragment DNA into a preselected, transcriptionally active locus in the rat genome. The webinar also discusses the advantages of generating transgenic rat models using this technology and the various applications that these rat models can be used in.
Highlights of the talk:
- Introduction to TARGATT™ integrase based technology
- How TARGATT™ technology is used to generate large fragment knock-in animal models
- Advantages of using TARGATT™ for site-specific knock-in compared to other gene editing technologies
- How we generate transgenic rat models using the TARGATT™ platform
TARGATT™ and CRISPR/Cas9 modified induced pluripotent stem cells (iPSCs) for in vitro genetic disease modeling (December 2015)
Applied StemCell is one of the prominent providers of gene editing services to generate transgenic animal and cellular models for researchers in academia and industries. This recorded presentation showcases Applied StemCell's achievements with using CRISPR/ Cas9 and its proprietary TARGATT™ gene editing technologies to modify induced Pluripotent Stem Cells (iPSCs). The webinar also explains the need for better models of human diseases and the advantages of using of genetically engineered iPSCs as in vitro models for genetic disease modeling. With examples and case studies, we describe how we optimize protocols to improve efficiency and are able to provide high quality service for generating iPSC disease models.
Highlights of this talk:
- Current trends in genetically modified iPSCs as disease models
- ASC's complementary technology platforms (CRISPR & TARGATT™) used for generating site-specific, genetically modified iPSC models
- Advantages of using genetically modified iPSCs and bottlenecks in gene editing of iPSCs
- ASC's upgraded protocols for high efficiency gene editing in iPSCs
- Examples of gene modification from patient/ healthy individual derived iPSCs