Molecular & Virology Services

Transcription

1 Molecular & Virology Services The laboratory staff at MQA is composed of experienced PhD level scientists whose expertise encompasses the disciplines of microbiology, molecular biology, and biochemistry. Our scientists also have years of experience with projects focusing on assay development, assay validation, and experimental studies. With this level of research experience, the scientists at MQA have developed the capability to solve research problems as well as perform routine analyses. They are also skilled at communicating with clients to make sure any questions are addressed and that the project runs smoothly. MQA laboratory scientists realize the importance of meeting the client s needs and focus their efforts on performing quality work while completing their projects on time. Our laboratory has been granted ISO accreditation by American Association for Laboratory Accreditation (A2LA) and is compliant with GxP FDA requirements. Cloning and Expression Systems Custom Vector Design, Production, and Amplification Research grade and fast turnaround times Cell Culture Services Mycoplasma Detection and Authentication by Isozyme Analysis Cell Line Production both Primary and Stable Expressors (Knock- Down or Over-Expression and constitutive or inducible promoter) Cytotoxicity, Proliferation, and Potency Bioassays Migration, Invasion, and Chemotaxis Testing Virus Titering and Production Services Virus Production (HIV, FeLV, Parainfluenza, Reovirus, Herpes, Polio, Others), Pseudotyped viruses (Retroviral, Adeno-viral AAV, or custom made-to-order) Adventitious agent screening and validation of viral decontamination Virucide Services: Activity, Reduction, or Surrogate Virus Testing Retroviral Detection by: S + L - Assay, TEM, Co-Cultivation, RT-Detection, and PERT Molecular Based Assay Services Protein Detection, Quantitation and Assay Development (ELISA, Western blotting, etc.) PCR Based Services: Reverse-Transcriptase and Teal-Time PCR assays qpcr for Pathogen Detection (Viral, Bacterial, and Mycoplasma)

2 Cloning and Expression Systems Molecular cloning is not always straightforward to perform and can be very time consuming. Save time and effort by outsourcing your molecular biology project to MQA. Together, we will help to define a customized strategy to meet your requirements. MQA s Molecular Cloning Services Include: Custom vector construction and cloning genes (full-length cdna) from total RNA or cdna library, PCR product, oligos/adapters, microdna or RNA Sub-cloning Vector Expression Optimization Expression Cloning with Retroviral Vectors Plasmid Amplification and Isolation Plasmid DNA preparation services have a flexible scale from MidiPrep (100 μg) up to gram level Research Grade: For typical molecular biology research Endotoxin Free Grade: Recommended for effective cell transfection, gene vaccine and protein manufacturing, and animal studies Advantages of MQA Cloning Services: Project designed by highly experienced molecular biologists to meet your requirements Progress report from dedicated project manager to keep you informed Broad range of services (sequencing, DNA preparation, and site-directed mutagenesis) Savings of your energy and time so that you can spend more time on your creative research Modularity: Each project is divided into modules with go/no go steps for more flexibility and security Vigorous Quality Controls and Standard Operating Procedures (SOPs)

3 Cell Culture Services Cell cultures represent a standard in vitro model in biomedical research. Without the need of using laboratory animals, they allow for the complex study of cellular mechanisms, both under physiological, and experimental conditions. Their application ranges from the study of metabolic routes in basic research, to production of recombinant proteins in the biotech or pharmaceutical industries. The techniques of working with cell cultures have been well established and at MQA, we have succeeded in establishing and standardizing a number of in vitro methodologies, which are now offered through our molecular services division. Mycoplasma Detection Services We provide testing of eukaryotic cell lines for the presence of all mycoplasma species from the culture media in a very short time. Mycoplasmas are the smallest and simplest prokaryotes. They are causal agents of numerous diseases of animals and humans and may also cause annoying contaminations in eukaryotic cell cultures. The presence of mycoplasmas in cellular lines may significantly influence the results of experiments because mycoplasmas may affect: 1) the cell growth rate which induces morphological changes which induces chromosomal aberrations, 2) the amino and nucleic acids metabolism inducing cell transformation, 3) molecular transport through the cell membrane. Mycoplasma Testing: For Human Vaccines and Biologicals Compliant with 21 CFR Multi-Media Direct Culture Method with Fluorochrome Staining Assay: This test consists of a multi-media Direct Culture method using three different mycoplasma media formulations combined with an Indicator Cell Culture procedure. The Indicator Cell Culture procedure with DNA fluorochrome assay enhances the detection of non-cultivable mycoplasma species. This is the best test for detecting mycoplasma contamination in cell cultures and is a GMP service. DNA Fluorochrome Staining Assay with Indicator Cell Line: This DNA fluorochrome assay with inoculation on an indicator cell line enhances the level of assay sensitivity by reducing background from genetically unstable cell lines (e.g. hybridomas, etc.) and amplifies the titer of mycoplasma contaminants. Developed for the detection of mycoplasma contamination in cell cultures and related samples, this test provides reliable and timely results.

4 PCR Based Detection Assay: Incomparably the fastest and easiest of all the tests done at MQA. In this assay, cellular particles of the cell culture supernatant are isolated by centrifugation and the DNA is separated and purified. Aliquots are used in PCR reactions with a primer mixture for the amplification of sequences which occur in the conserved 16S rdna regions of almost all mycoplasma species listed so far in publicly available sequence databases. After contamination, a bp DNA fragment (depending on the mycoplasma species) is visible in the ethidium bromide stained agarose gel. As an internal control, we coamplify a ca. 975 bp DNA fragment in parallel to verify successful PCR amplification regarding sensitivity and absence of Taq polymerase inhibitors. Other Mycoplasma Services Include: Reagent Standards Mycoplasma DNA for Assay Development. Positive and Negative Control Slides for in-house development MQA specially coated slides for your lab to use at your convenience. Simply add your cell culture sample, fix, and dry. Return the (hazard-free) slides to our laboratory for DNA fluorochrome staining and diagnosis. Primary cell line generation: Primary cells are cells straight from the tissue with no passages. A primary cell culture may be composed of mixtures of cell types. Frequently the some of the cells may survive without proliferating and will therefore be lost in the increasing population of those which are able to multiply in the conditions supplied in vitro. Cells from explants may sometimes be converted to cell lines by passage. These may continue to proliferate for a number of cell generations. MQA also offers cells for use of primary fibroblasts as feeder layers for the growth of some embryonic stem cell types. At MQA our scientists can prepare primary cell lines from most tissues using our standard protocols. If you would like to implement a custom project, we would be happy to discuss your project specifications with you.

5 Cytotoxicity/Growth Assays: Testing by MTT, XTT, or any customized approach, such as Alamar blue, is based on the ability of metabolically active cells to convert special dyes into measurable flurophores such as yellow tetrazolium salt, XTT, into orange formasan. Complex rinsing is thus avoided, which is necessary, for example, in the older MTT method. This guarantees high accuracy of the XTT test and allows for fast processing of the data obtained. The bioassay may also be used to establish relative, cytotoxicity of agents within various chemical classes. We can establish baseline data for predicting the toxicity of related novel agents by comparing such baseline data with known in-vivo toxicity. The assay is simple to perform since the indicator is water soluble, thus eliminating the washing/fixing and extraction steps required in other commonly used cell proliferation assays. Authentification by Isozyme Assay: One of the major problems in cell culturing is the misidentification or cross-contamination of authentic continuous cell lines. Confirmation of purity and identity of cell cultures is a necessary step in the production of biotherapeutics. Working with several cell lines in the same facility can result in cross contamination while mislabeling of cultures can cause misidentification. The method is based on the isoelectric separation of a specific set of intracellular enzymes which can be used to distinguish between human, murine, or other mammalian cell lines. Stable Cell Line Expression: For either over or knock-down by shrna. Stable cell lines with specific gene over-expression or knock-down are very helpful in gene function analysis, target discovery, target validation, assay development, and compound screening. MQA s team of experienced scientists will work with you to establish the system to generate your particular cell line for your specific application. Since long-term suppression using pol III shrnas can be problematic especially for studies of genes that are critical to cell proliferation and differentiation, we provide not only constitutive but also conditional knock-down. MQA Service will provide you with: Your choice of a constitutive or inducible promoter, a tag, a selection marker and a fluorescent marker. Either you provide target templates or we design, synthesize, or obtain it from a cdna collection. Clone your gene or shrna into our lentiviral vector. Generate lentivirus.

6 Transduce the cell line of interest. Select the stably transduced, high expression cells. Validate the genomic integration of the target by genomic PCR. Validate the high-expression clone by Western Blot (if applicable). Produce two cryogenic preserved vials of stable cells and a final report. Why use MQA Cell Culture Services: Safe-to-use (self-inactivating) lentiviral particles can deliver your gene into a wide range of cell lines including non-dividing, primary or stem cells. Engineered in-house lentiviral vector for highly efficient gene integration into cell genome. Our lentivectors and standard C-type retroviral vectors have the strongest promoter for the highest protein expression in mammalian cells. You can choose to have an inducible* or constitutive promoter. Our experts have years of experience in lentiviral cloning and expression. Fast turnaround time. The best price and the best quality in its class Other Cell Culture Based Services: Cell viability (cytotoxicity) Cell proliferation Apoptosis Cell adhesion Cell migration Cell invasion Cell shape change Chemotaxis Angiogenesis Soft Agar Colony Formation Assay Cell culture and expansion of various primary and cancer cells Receptor binding

7 Virology Services MQA Laboratories offers a variety of virology and cell based assays. We can customize these services to meet the needs of our customers. We are continuously adding to our portfolio of services and organisms which include: Growth and Tittering of viral stocks Generation of viral vectors Growth of Pseudotyped viruses (Retroviral, Adeno-viral AAV or Custom made to order) o Virucidal Activity Studies o Disinfectant Virucidal Efficacy Tests o Virucidal Surrogate Efficacy Tests o Adventitious agent screening and validation of viral decontamination. o Specific Viruses Bovine Viral Assay (according to 9 CFR 113) Porcine Viral Assay (according to 9 CFR 113) o Retrovirus Assays XC Assay for Ecotropic Retroviruses S+ L- Assay for Xenotropic Retroviruses Electron Microscopy for Microscopy for Evaluation of Cell Banks and Culture Harvests Co-Cultivation o Antibody Production Mouse Antibody Production (MAP) Hamster Antibody Production (HAP) Rat Antibody Production (RAP) Examples of viruses available: o HIV/FIV o Herpes o Influenza H1N1 o Polio o Parainfluenza o Norovirus o Reo o Others available upon request

8 Molecular Based Assay Services MQA offers a wide range of assay design and development services. Functional assays are designed, validated and customized to the needs of our pharmaceutical and biotechnology industry clients. These assays are aimed at assessing and monitoring the efficacy, tolerability and safety parameters of candidate therapeutics and prophylactics in cancer, infectious disease, autoimmunity and transplantation. Assays are derived from pertinent scientific literature or are directly implemented from already developed procedures. GLP and GMP assays can be validated based on client needs. Full custom assay development available based on a target of your choice. Assay Services Cell viability (cytotoxicity) Cell proliferation Apoptosis Cell adhesion Cell migration Cell invasion Chemotaxis Angiogenesis Soft Agar Colony Formation Assay Cell culture and expansion of various primary and cancer cells Receptor binding Cell shape change Endotoxin measurement (LAL assay) Cellular uptake of FITC-labeled drug ELISA & Western Blot development and validation of kits Serology work including development and validation of ELISAs Method development and validation, following U.S. Food and Drug Administration guidelines DNA transfection and protein detection (ELISA and Western Blot) TF-1 cell proliferation assays (Alamar Blue) MTT-based cell proliferation assays (Mitochondrial Function) Apoptosis assay Cytotoxicity Assays perform in compliance with USP <87> and ISO

9 Chemiluminescent DNA Replication Apoptosis Assay Development Like any new project, additional work may be needed to prepare the assay(s) for high-throughput screening. Assay development is defined broadly, from simple technology transfer, to complete de novo development, depending on the client's target and the assay's stage of development. MQA will apply the best technologies for measuring and screening of biomolecular interactions and functions of cells to reduce the expense and to improve the efficiency of the screening process. Assay Options: Custom assay development based on a target of your choice Rapid, testing of lead compound analogue libraries or screening hits for multi-parametric activity profiles (primary assay potency, in vitro, selectivity) Multiple assay platforms/detection methods Red Cell Adherence Western Blot Recombinant Immunoblot Assay PCR Based Assays PCR is a powerful, highly sensitive technique that can be used to quantitate gene expression, determine gene copy number, detect SNPs, and detect DNA from viral and bacterial microorganisms. We utilize the Applied Biosystems platform for Sequence Detection to perform qpcr experiments. PCR Services Include: Support of both SybrGreen and Taqman reaction chemistries Primer and probe design services Mycoplasma testing by PCR RT-PCR Development of Standards for qpcr PCR assay design, development and validation Reverse-transcriptase and real-time PCR assays qpcr for MMV and other Infectious Agents