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Abstract: Dopaminergic neurons play significant roles in motor, reward, and motivational behavior related circuits throughout the brain. To date, there are few continuous in vitro models available to laboratories in research, industry, and academia for studies related to basic dopaminergic cell biology or high throughput screening. Here, we propose the use of a human model system, LUHMES cells (ATCC® CRL-2927™), to study dopaminergic neuron cell biology and Parkinson’s disease. LUHMES cells are neuronal precursors derived from fetal ventral mesencephalon. Neuronal differentiation is governed by the termination of v-myc expression using low levels of tetracycline. During our characterization, we found that tetracycline induced differentiation resulted in consistent neurite outgrowth in LUHMES cells within two to four hours. One day post differentiation, cells displayed similar morphology, with several long processes protruding from the cell soma. Growth cones were often observed in early differentiated cultures. Immunocytochemistry in early differentiated cultures (Days in vitro, DIV 2-3) revealed low level expression of tyrosine hydroxylase; however, these levels were increased significantly by 7 DIV with many neurons expressing tyrosine hydroxylase. We also investigated dopamine transporter expression. Differentiated LUHMES cultures were positive for neuronal markers such as bIII tubulin and devoid of expression of traditional glial markers including GFAP and IBA-1. Both undifferentiated and differentiated LUHMES cells were easily transfected using basic eGFP constructs, although greater efficiencies were observed with the use of viral constructs. In summary, LUHMES cells are a suitable and robust in vitro model system for studying dopaminergic neuron cell biology and mechanisms underlying Parkinson’s disease.

ATCC is among the world’s leading biological resource centers, supporting both the academic and commercial research and development communities. The organization houses and supplies a wide range of biological materials and research tools, including continuous cell lines, primary cells, and hTERT-immortalized cell lines, as well as bacteria, viruses, fungi, and protists. In addition, ATCC develops and supplies microbial and tumor cell panels, certified reference materials, culture media and sera, and other research materials. This module examines the use of ATCC materials in two areas: molecular-based assays for cancer detection and analysis; and assay development for infectious diseases.

Genetic sequencing advances and molecular diagnostics are fostering the development of personalized medicine research and care. This has amplified the need for reliable biological certified reference materials for use as controls in cancer research and elsewhere. ATCC has developed a host of research materials, including authenticated cell lines and cell derivatives to support molecular-based assay development. Examples include cell lines with well-characterized mutations relevant to cancer study and purified genomic DNA preparations with key oncological biomarkers.

Several key aspects of infectious disease assay development are aided by well-characterized and authenticated biological materials. These assay-development activities include sourcing organisms with known traits and relevant genes, establishing inclusivity/exclusivity parameters, and determining sensitivity and limits of detection. The ATCC portfolio contains an extensive collection of microbial cultures and genetic material designed to support assay development in infectious disease and public health research. Among the tools are microbial cultures of known source of isolation, fully sequenced strains, cultures with clinically relevant phenotypic characteristics, and synthetic nucleic acids with clinically relevant gene sequences. ATCC also holds cultures grouped by toxin production, serotype, and drug-resistance. All of these materials and tools are developed and handled in conditions that have been certified and accredited to four relevant ISO standards.

Abstract: Large-scale cancer genome programs have generated a rich data set comprising genetic abnormalities observed in thousands of clinical patient tumors, which provides a major opportunity for the molecular detection of cancer. However, the lack of controls for molecular tests has been a challenge. Because of the reproducible nature of the cell lines, genomic DNAs of fully characterized and authenticated cell lines provide a solution.

Genomic DNAs were extracted from over 70 commonly used human cancer cell lines derived from the breast, lung, colon, and pancreas, as well as hematopoietic and lymphoid tissue. Cancer gene mutations were identified by next-generation sequencing. Gene copy number changes were analyzed using the qBiomarker Copy Number PCR Assays kit (QIAGEN). Moreover, the selected cell lines were analyzed by quantitative polymerase chain reaction (qPCR), Western blot, and immunofluorescence (IF) staining to verify gene and protein expression mutation.

Abstract: Positive controls are essential for establishing assay performance and equipment efficacy. Yet, some food testing laboratories refrain from using bacterial strains as positive controls for fear of cross-contaminating their samples. Under the Food and Drug Administration (FDA) Food Safety Modernization Act, laboratories face an increasing number of regulations to expand testing for objectionable organisms. Control strains with unique, easily detectable traits which distinguish positive control strains from actual food contaminants can help differentiate true contamination from control strain cross-contamination.

In this study, we introduced shuttle vectors encoding either green fluorescent protein (GFP, Life Technologies) or NanoLuc® (Promega) into Escherichia coli strains, including Shiga toxin-producing O157 (stx1+, stx2+, eaeA+) and non-Shiga toxin-producing O157 (stx1-, stx2-, eaeA-), for use in food pathogen detection assays. Both reporters can be easily visualized without specialized detection equipment; GFP fluoresces when excited by UV light, while bacteria engineered with NanoLuc emit a strong light signal in the presence of a chemical substrate. Upon establishing the detectability of NanoLuc in the E. coli O157 strains, the reporter was transformed into the “Big Six” non-O157 E. coli strains (serogroups: O26, O45, O103, O111, O121, and O145) for use as reporter-labeled positive controls.

Abstract: Fluorescent proteins, such as green fluorescent protein (GFP), have diverse applications in the basic and applied sciences. While GFP has been frequently used in eukaryotic systems, its applications have been limited in microorganisms due to a lack of broad-range molecular tools. In this study, we have developed a vector to express GFP in pathogenic bacteria for use in bacterial pathogenesis and pathogen-host studies. A shuttle vector encoding the GFP variant mut31 (pUCP18-MCSgfpmut3) was generated and successfully transformed into various Gram-negative opportunistic pathogens from the ATCC collection, including: Escherichia coli (ATCC® 25922™), Salmonella enterica (ATCC® 14028™), Shigella flexneri (ATCC® 12022™), Pseudomonas aeruginosa (ATCC® 10145™), and the P. aeruginosa type strain PAO1 (ATCC® 15692™). P. aeruginosa was used as a model to test the characteristics of the vector and sensitivity of detection using a fluorescence plate reader, microscopy, flow cytometry, and in vivo imaging systems.

Abstract: Influenza is one of the most significant causes of acute respiratory infection worldwide. Rapid diagnostic tests for highly contagious pathogens, such as Influenza, are essential for decreasing the public health impact of emerging infectious diseases and bioterrorism agents. However, these tests require positive controls that are not always readily available. Consequently, if worldwide public health laboratories are unable to meet the costly regulations required for the import, transfer, and safe use of pathogens used as controls, then critical diagnostic, surveillance, and epidemiological information could be missed.

The use of in vitro synthesized viral RNA as a control would provide essential equivalency standards that would be accessible to any laboratory performing quantitative RT-PCR tests. Synthetic RNA controls are particularly useful for laboratories which lack appropriate biosafety containment facilities for propagating a particular pathogenic virus, or have difficulty gaining access to the organism in question due to international tightening of both import and export controls.

Abstract: Dengue fever is an acute illness caused by any one of four serotypes (1-4) of genetically related dengue viruses (DENV), with an estimated 390 million cases reported annually. Currently, quantitative RT-PCR (qRT-PCR) is the preferred method for the detection and quantification of DENV in clinical diagnostics and epidemiological surveillance. The accuracy of a qRT-PCR assay relies on the generation of a standard curve using a positive control with a known viral genome concentration.

Native DENV RNA can be used as a standard for these assays; however, the full-length dengue viral RNA is on the Commerce Control List and requires a permit from the US Department of Commerce for international shipment. To make DENV RNA standards more accessible, ATCC has developed four synthetic molecular standards that represent DENV serotypes 1-4. Each standard contains short fragments from the capsid, membrane, and envelope genes of the DENV genome, as well as target regions encompassing the primer sequences from numerous published RT-PCR assays, including the DENV-1-4 Real-Time RT-PCR Assay developed by the CDC1. The synthetic RNA standards were quantified by Droplet Digital™ PCR (ddPCR™) in order to package precise copies of RNA. Moreover, given the inherent labile nature of RNA, a stabilization matrix was added to the quantitated RNA preparation. As compared to native RNA, these synthetic standards are easier to use as controls for qRT-PCR assays, exhibit less variability, have a longer shelf life, eliminate the need to culture viruses and can be used under BSL-1 conditions. Further, this synthetic quantitative RNA approach can be extended to other pathogenic viruses which are unculturable or need to be grown in a high-containment facility.

In the following proof-of-concept study, we amplified the synthetic molecular standards with the published primers from the CDC assay1 and Waggoner et al2, and used the generated standard curves to quantify viral RNA extracted from various DENV strains.

ATCC Molecular Signature Panels

Powerful tools for the genomics age

12/14/2013 — 12/18/2013

ATCC molecular signature panels focus on key components of cell signaling pathways such as EGFR, AKT, PI3K, PTEN, or p53. This section will describe how we generated these panels using authenticated cell lines containing critical gene copy number changes and site mutations, as well as how each panel was experimentally validated for genetic alterations, protein expression, and cell functionality.

Transfex

for hard to transfect cells

12/14/2013 — 12/18/2013

ATCC offers a superior lipid-based transfection reagent (TransfeX) that can be used to transfect difficult cell types, like primary and stem cells, and uncomplicated continuous cell lines. In this section, we will show how TransfeX provides higher transfection efficiency and lower cytotoxicity than other commercially available transfection reagents. We will also describe the HEKPlus Expression System for protein expression.

Induced Pluripotent and Mesenchymal Stem Cells

Cells with a lot of potential

12/14/2013 — 12/18/2013

ATCC has a wide selection of induced pluripotent and mesenchymal stem cells along with an array of associated culture media and reagents. This section will provide an overview of the ATCC stem cell collection and describe how these resources can be used in cell biology studies.