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Validation of Recovery and Purification Processes
Published in James Agalloco, Phil DeSantis, Anthony Grilli, Anthony Pavell, Handbook of Validation in Pharmaceutical Processes, 2021
For proteins produced by recombinant DNA technology in mammalian or human cells (e.g., CHO, BHK, C127, HEK-293, and hybridomas), virus removal and inactivation validation include model viruses possessing a range of biophysical and structural features.68 The viruses used almost always include Xenotropic Murine Leukemia virus (a model retrovirus),69 Minute Virus of Mouse, and two or three additional viruses that are enveloped and non-enveloped and DNA or RNA viruses with different diameters and geometries. Examples of additional DNA viruses used in viral clearance studies include Herpes Simplex 1 (enveloped) and SV-40 (non-enveloped) and RNA viruses include Sabin Type I Polio (non-enveloped) and Influenza Type A (enveloped). Additional viruses commonly used in viral clearance studies include Infectious Bovine Rhinotracheitis Virus, Reovirus Type III, Epstein-bar Virus (hybridomas), and Pseudorabies Virus. When choosing an appropriate challenge virus, preference should be given to those viruses that display a significant resistance to physical and/or chemical agents.
Saccharomyces cerevisiae
Published in Shakeel Ahmed, Aisverya Soundararajan, Pullulan, 2020
To detect sense and antisense expression, reverse transcriptase (RT) was employed using a Molony–Murine Leukemia Virus and other reagents supplied with the Retroscript First Strand Synthesis Kit for RT-PCR (Ambion). For RAPD-PCR of the cDNA, Supertaq Taq Polymerase (Ambion) optimized for the Retroscript kit was utilized, and the annealing temperature for the O-AB04 primer was used. Appropriate negative controls (supplied with kit, S. cerevisiae not transformed, S. cerevisiae antisense, water as a blank, etc.), were included in each process. Two-step RT-PCR was performed according to the Ambion manual, in which 2 µg of template RNA was used. The following parameters were programmed into the thermocycler: 1 cycle of 95°C for 5 min, 30 cycles of 94°C for 30 s, 30°C for 30 s, and 72°C for 1 min, and a final cycle of 72°C for 5 min.
Enzymes Used for Recombinant DMA Technology Produced by Recombinant Microbes
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
The Mo-MLV reverse transcriptase is the product of the Molony murine leukemia virus pol gene and consists of a single polypeptide chain (Mr 84,000). This enzyme has an RNA-dependent DNA polymerase activity and a RNase H activity [44]. The enzyme is used to synthesize cDNA from mRNA. The major problems on cDNA synthesis are caused by the RNase H activity of reverse transcriptase. At the beginning of cDNA synthesis, the hybrids formed between primer and template mRNA are substrates for RNase H. Thus, there is a competition between degradation of the template mRNA and initiation of DNA synthesis. In addition, RNase H can cleave the template near the 3' terminus of the growing DNA strand if there is a reverse transcriptase pause during synthesis. Attempts to separate the active centers of RNA-dependent DNA polymerase and RNase H activity by proteolysis have not succeeded.
Physiological and molecular responses of the root system of indica–japonica intersubspecific tetraploid rice seedlings to ion beam stress
Published in Radiation Effects and Defects in Solids, 2021
Yaqin Huang, Qunce Huang, Yue Yin, Zhen Jiao
Total RNA was isolated from 0.10 g of 10-day-old fresh seedling roots from non-irradiated and irradiated seeds exposed to ion beams using RNAiso TM Plus (TaKaRa, Japan). The complementary DNA (cDNA) of rice was synthesized using Moloney murine leukemia virus reverse transcriptase (Invitrogen, USA) following the manufacturer’s instructions. The cDNA samples were used as templates for the quantitative reverse transcription PCR (qRT-PCR), which was performed using a Mastercycler EP Realplex thermal cycler (Eppendorf, Germany). An SYBR-specific fluorophore was utilized as a quantitative tool to monitor the reactions, and the DyNAmoTM SYBR Green qPCR kit was used for the reagents. The following standard thermal profile was used for all the PCRs: pre-denaturation at 95 °C for 2 min, denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and prolongation at 72°C for 20 s with 40 cycles. Data were analyzed using Realplex 2.2 software (Eppendorf, Germany) to calculate cycle threshold values. Using the OsActin gene as the internal reference, the relative expression of the target and internal reference genes was analyzed using the comparative Ct (2−ΔΔCt) method. The primers of the genes were synthesized by Shanghai Meiji Biomedical Technology Co., Ltd.
Licochalcone A, a licorice flavonoid: antioxidant, cytotoxic, genotoxic, and chemopreventive potential
Published in Journal of Toxicology and Environmental Health, Part A, 2020
Karoline Soares de Freitas, Iara Silva Squarisi, Nathália Oliveira Acésio, Heloiza Diniz Nicolella, Saulo Duarte Ozelin, Matheus Reis Santos de Melo, Ana Paula Prado Guissone, Gabriela Fernandes, Lívia Mara Silva, Ademar Alves da Silva Filho, Denise Crispim Tavares
MMS also produces depletion of reduced glutathione (GSH) and associated enzymes in mammalian cells. These enzymes are important for the antioxidant cell defenses and depletion results in accumulation of reactive oxygen species (ROS) generated as byproducts of normal cellular function (Coles and Kadlubar 2003; Ginsberg et al. 2009; Lee et al. 2007; Liu, Lightfoot, and Stevens 1996; Lu 2013; Mizumoto, Glascott, and Farber 1993; Moine et al. 2018; Trézl et al. 1987). Lv et al. (2015) noted that LicoA significantly inhibited tert-butyl hydroperoxide-induced ROS generation and decreased GSH depletion by enhancing the expression of the glutamate-cysteine ligase modifier subunit and glutamate-cysteine ligase catalytic subunit genes in RAW 264.7 cells (Abelson murine leukemia virus-induced tumor). These genes are involved in GSH biosynthesis (Lu 2013). Therefore, in addition to its free radical-scavenging activity and upregulation of HO-1 and Nrf2 expression, the antioxidant potential of LicoA may also be attributed to its effect of upregulating GSH biosynthesis, which might have contributed to protection against MMS-induced genotoxicity.
HEMA and alginate-based chondrogenic semi-interpenetrated hydrogels: synthesis and biological characterization
Published in Journal of Biomaterials Science, Polymer Edition, 2020
María Luz Torres, Tamara Gisela Oberti, Juan Manuel Fernández
For RT–PCR studies, total ribonucleic acid (RNA) was isolated from cells grown on scaffolds for 10 days. TRIZOL reagent was used as indicated by the manufacturer (Invitrogen, Argentina). Expression of chondrocytic and osteogenic markers was performed by semi-quantitative RT–PCR using Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) (Invitrogen, Argentina). Expressions of all markers were normalized to β-actin (housekeeping gene). Specific primers for all markers were designed from The National Center for Biotechnology Information (NCBI) sequence data using CLC Genomics Workbench software (QIAGEN) (Table 2) and synthesized by Macrogen (Seoul, Republic of Korea). After separation of RT-PCR products by agarose gel electrophoresis with GelRed, their corresponding band intensities were quantified using the gel plugin of MBF_ImageJ program.