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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.
Production of VNPs, VLPs, and Chimeras
Published in Nicole F Steinmetz, Marianne Manchester, Viral Nanoparticles, 2019
Nicole F Steinmetz, Marianne Manchester
Genetic engineering of VNPs refers to the manipulation of the genome, which results in modifications on the protein level. All plant viruses currently in use for nanotechnology applications have RNA genomes. The genomes have been sequenced, and the genetic information is available at the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). To perform genetic modifications, a cDNA copy of the genome is required. The cDNA is the complementary strand of the genome RNA, which can be synthesized by reverse transcription. The cDNA can then be amplified as double-stranded DNA using PCR techniques and inserted into a cloning or expression vector. At this stage, any standard cloning or mutagenesis procedure can be applied in order to introduce the desired modification. For detailed background information on cloning techniques, the reader is referred to textbooks in the fields of molecular biology and biochemistry.
Mechanostimulation in Bone and Tendon Tissue Engineering
Published in Jiro Nagatomi, Eno Essien Ebong, Mechanobiology Handbook, 2018
Samuel B. VanGordon, Warren Yates, Vassilios I. Sikavitsas
PLLA scaffolds are removed from RNAlater®. Scaffolds were then broken down and RNA isolated using RNAqueous®-4PCR Kit (Ambion) in accordance with the manufacturer's directions. cDNA was then synthesized from RNA using TaqMan® Reverse Transcription Reagents (Applied Biosystems, Austin, TX) in accordance to manufacturer's directions using random hexamers as the primers for reverse transcription. Gene expression levels of collagen I (Coll-a1), collagen III (Coll-a3), and Cox-2) were relatively quantified using a Power SYBR® Green PCR Master Mix kit (Applied Biosystems) using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as reference. Primer sequences were as follows: Coll-a1 forward 5′-GGA GAG TAC TGG ATC GAC CCT AAC-3′ and reverse 5′-CTG ACC TGT CTC CAT GTT GCA-3′; Coll-a3 forward 5′-CAG CTG GCC TTC CTC AGA CTT-3′ and reverse 5′-GCT GTT TTT GCA GTG GTA TGT AAT GT-3′; Cox-2 forward 5′-TAC TGT GTA GCT CCC CTT CG-3′ and reverse 5′-TGC CCA GAA CTA CCC ACTA A-3′; GAPDH forward 5′-AAC TCC CTC AAG ATT GTC AGC AA-3′ and reverse 5′-GTG GTC ATG AGC CCT TCC A-3′. Quantitative real-time PCR was conducted as follows: 10 min at 25°C, followed by 45 cycles of amplification where each cycle consisted of 15 s at 95°C followed by 1 min at 60°C. Relative gene expression (compared to GAPDH) was analyzed using the ΔΔCT method [164].
Artemisia vulgaris essential oil nanoemulsions (AVEO-NE), a novel anti-angiogenic agent and safe apoptosis inducer in MCF-7 human cancer cells
Published in Inorganic and Nano-Metal Chemistry, 2022
Mahjoubeh Irani, Masoud Homayouni Tabrizi, Touran Ardalan, Toktam Nosrat
The expression of genes similar to the research conducted by Khatamian et al. was examined by qPCR method.[25] First, the MCF7 cells were seeded in a 12.5 cm2 culture flask for 24 h and then treated with various doses of AVEO-NE (1, 2, and 4 µg/mL). After 48 h, the RNA content of cells in each group was extracted using the RNeasy mini kit (Qiagen, Hilden, Germany). Then RNA was used as a template for CDNA synthesis and complementary DNA was synthesized using Quantitect Reverse Transcription kit (Qiagen, Hilden, Germany). The target gene primer sets for Cas-9,[26] CAT, SOD, and VEGF[25] are given in Table 1. The GAPDH gene[27] was defined as an internal control gene (Table 1). In order to perform the reaction, a mixture with a volume of 20 μL including 10 μL of Syber Green, 2 μL of specific primer, 1 μL of CDNA, and 7 μL of DW was prepared and analyzed by CFX-96 Biorad.
Methacrylated pullulan/polyethylene (glycol) diacrylate composite hydrogel for cartilage tissue engineering
Published in Journal of Biomaterials Science, Polymer Edition, 2021
Xiaoping Qin, Rui He, Hao Chen, Dejie Fu, Yang Peng, Shuo Meng, Cheng Chen, Liu Yang
For gene expression analysis, MSCs-encapsulated hydrogels (n = 3) at 7 and 14 days were collected and ground with liquid nitrogen. First, total RNA was extracted from MSCs-encapsulated hydrogels using the Trizol (Roche, Basel, Switzerland), and RNA concentration was determined using a NanoDrop-2000 spectrophotometer (Thermo Scientific). Subsequently, RNA was reverse transcribed into cDNA using the Transcriptor cDNA Synth. kit 2 (Roche). Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was carried out with an initial incubation at 95 °C for 2 min, followed by 45 cycles of denaturation (95 °C, 10 s), annealing (specific for each gene, 25 s) and extension (72 °C, 30 s) following the FS Essential DNA Green Master (Roche). Cartilage markers collagen type II (COL2A1) and aggrecan (ACAN), early chondrogenic transcription factor SOX9, and hypertrophic marker collagen type X (COL10A1) genes were quantified. The obtained results were analyzed according to the 2-△△CT method and normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences (Sangon Biotech, Shanghai, China) used for RT-PCR were listed in Table 1.