GHP April 2017

14 GHP / April 2017 , lines engineered to overexpress a biomolecule of interest is extremely valuable for research studies exploring gene function and drug screening. Furthermore, preclinical studies involving mammalian cell lines allows a smoother transition into clinical studies. However, although extremely useful for applications in research, production of stable cell lines and primary cells can be resource draining with respect to cost, labor, and time. Our lab offers several stable cell line engineering services with the following properties: protein-overexpression, RNAi knockdowns, and reporter gene expressing (luciferase, GFP, RFP, YFP). Our Scientists have ample experience in stable cell line generation and we offer more than 150 different cancer cell lines. We develop all stable cell lines concordant with client specifications and accomplish this in a 28 day timeframe. Additionally, we can produce multiple clonal cell lines having varying levels of expression for a biomolecule of interest (low, medium, or high). Our standard cell line generation services include transfection of plasmid DNA (10 – 20 μ g), drug selection of clonal cells, colony picking, generation of a stable cell line, expression and functional screening (a minimum of 10 passages), and final validation of construct expression by qRT-PCR and/or Western Blot analysis. This two-method validation of stable cell line development ensures that the gene of interest is both integrated into the genome and concurrently being expressed. Cell Banking Services Altogen Labs also offers GLP-compliant cell-banking services. To ensure indefinite longevity to cells, cryogenic storage in liquid nitrogen at -196 o C is recognized as the best possible practice. Preserving the integrity of a single cell line in culture for the purposes of research and manufacturing can be troublesome due to factors like sample contamination by microbial species, target phenotype change, genetic drift, and uncertainty with respect to the finite life span of a cell line. Access to a cell bank allows the client to preserve consistency in research and manufacturing as this grants access to a cell reservoir of low passage number, free of microbial contamination, and phenotypically intact for study. Altogen Labs complies with regulatory measures to ensure that all procedures, practices, and facilities are up to current GLP standards, and ensures that all cell lines are free of bacterial and mycoplasma contamination. Our protocols use combinations of cryoprotectants and cooling steps to ensure successful cryopreservation of biological materials, cells, and tissues. Altogen Labs banks stable cell lines, cells for transfusion, umbilical cord blood cells, stem cells, and tumor/histological cells. Altogen Labs offers our clients long term cell storage in a secure, controlled, and monitored environment. All cell banking work is performed under certified laminar flow hoods, and cells are grown in calibrated incubators with ultraviolet (UV) irradiated air and water. Replicate cell samples are taken and stored in separate freezers to mitigate the risk of temperature failure. The freezer temperature and liquid nitrogen levels are monitored round the clock. Cryovials can be used to expand a cell population, or shipped on dry ice to any worldwide destination. RNAi Studies RNA interference (RNAi) is a specific, in vitro gene silencing tool employed for gene function studies in cultured cells. Targeted silencing of gene expression is achieved by the introduction of small- interfering RNA (siRNA) or microRNA (miRNA). An siRNA molecule is a double stranded oligonucleotide, 20-24 base pairs in length, having a hydroxylated, 2’ nucleotide overhang at each 3’ end and phosphorylated 5’ ends. Alternatively, a miRNA is a 22 nucleotide, single stranded RNA molecule that targets and binds to a complementary RNA sequence to yield a double stranded RNA molecule. Typical introduction of siRNAs or miRNAs into the cell via chemical transfection methods can transiently silence expression of target genes for three to seven days. Although useful, the in vitro RNAi experiment is limited in not allowing for gene function study in the organism altogether, as an in vivo study would. However, the in vivo study presents its own set of challenges in the form of RNA instability, inefficient delivery, incorrect biodistribution, and the intrinsic difficulty of targeting specific tissues. For these reasons, in vivo RNAi experiments can be costly and time consuming. In a typical gene function study, siRNAs and miRNAs are introduced by direct administration or via plasmid DNA and viral vectors, the latter needing cellular processing to become biochemically functional. Generally, the viral vector method is the most efficient in vivo, however, the vectors are relatively difficult to construct and run the risk of immunogenicity issues. Once administered, the biggest obstacle to an in vivo RNAi study is the specific delivery of the ribonucleotide to the target tissue. Achievement of this would require that the siRNA or miRNA has to endure degradation by endogenous nucleases, evade immune system detection, minimize off target effects, and ultimately, be endocytosed by the target tissue. Altogen Labs has developed RNAi methods for in vivo studies that prevent siRNA degradation in serum via chemical modification and liposome encapsulation. These modifications enhance delivery of the ribonucleotide to the target organ, and trigger significant, tissue-targeted functional effects. Chemical modifications of siRNA and miRNA in vivo include 2’MOE, FANA, 2’-Fluoro, and LNAs. Altogen Labs provides complete in vivo siRNA development services including in vitro siRNA testing in cell- based assays, encapsulation and tissue-targeted siRNA delivery into animal models (including mouse, rat, and xenograft cancer models). We also offer multiple siRNA administration routes for local and systemic delivery including intravenous, intraperitoneal, intranasal, intratumoral, intratracheal, intradermal, intramuscular, intrathecal, intracerebellar, and intravascular perfusion. Bioremediation Bioremediation is the process of using microbes, fungi, green plants, and their endogenous enzymes to restore a contaminated environment to a non-toxic or less toxic state 1 . Bioremediation is one of the biotechnology focus areas of Altogen Labs . Our team of scientists has applied techniques in biotechnology to develop patent-pending bioremediation products and services to offer to our clients. Our technology is based on harnessing and enhancing the ability of microorganisms found in soil and water to digest hydrocarbons, thus accelerating the conversion of pollutants to less-toxic or innocuous substances. Altogen Labs has developed methods for isolation, selection, and cultivation of highly efficient hydrocarbon-degrading microbes specific to the site of a particular spill. Our technology is scalable, and has been optimized to account for variables like temperature, soil conditions, and oil composition. Through our unique methods, we have developed a library of over 150 strains of natural oil-degrading bacteria. These microbes have been demonstrated in the laboratory and in field tests to be highly efficient for remediation of crude-oil and petroleum saturated earth. Our bioremediation products are fast, safe, and cost-effective for restoring large amounts of soil and liquids that have become hydrocarbon contaminated (oil, petroleum, pesticides, carbon tetrachloride, acetone, benzene,

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