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Use of Recombinant DNA Technology for Engineering Mammalian Cells to Produce Proteins
Published in Anthony S. Lubiniecki, Large-Scale Mammalian Cell Culture Technology, 2018
The 3′ end of eukaryotic mRNA is formed by polyandenylation, which involves cleavage of the precursor mRNA at a specific site and then polymerization of about 200 adenylate residues, poly (A), to the newly generated 3′ end (151). Removal of the polyadenylation site decreases expression up to 10-fold (152). Two sequences important for polyadenylation have been identified. The first is a highly conserved hexanucleotide AAUAAA, present 11–30 nucleotides upstream of most polyadenylation sites, which forms the recognition sequence for the cleavage and polyadenylation reaction. Deletions or point mutations in this sequence prevent the appearance of properly polyadenylated mRNA in vivo (153–156). There is also a requirement for a sequence downstream of the poly (A) site for efficient cleavage and polyadenylation (157, 158). A loose consensus sequence for this potential second element was identified as either a U-rich or a G + U-rich tract (159). However, removal of this sequence in some instances has no effect on the efficiency of polyadenylation (160) but may influence the position of 3′ processing (161). This sequence appears to be required for the formation of a precleavage complex (162).
Glossary of scientific and technical terms in bioengineering and biological engineering
Published in Megh R. Goyal, Scientific and Technical Terms in Bioengineering and Biological Engineering, 2018
Polyadenylation refers to post-transcriptional addition of a polyadenylic acid tail to the 3’ end of eukaryotic mRNAs. Also called poly-(A) tailing. The adenine-rich 3’ terminal segments is called a poly (A) tail.
High-level production and purification of bioactive recombinant human activin A in Chinese hamster ovary cells
Published in Preparative Biochemistry & Biotechnology, 2023
Changin Kim, Hyunjoo Kim, Jeong Soo Park, Jiwon Park, Jeongmin Oh, Jaeseung Yoon, Kwanghee Baek
For the construction of the rhActivin A expression plasmid, the nucleotide sequence encoding the human activin A preproprotein (INHBA, GeneBank Accession No. BC007858) synthesized by Bioneer Corporation (Korea) was cloned into the mammalian expression vector, pC(F)mEGM(R)-TA, which was reported to induce high expression of recombinant proteins in CHO cells.[23] The expression vector, pC(F)mEGM(R)-TA, contains cytotoxic serine protease-B(CSP-B) scaffold attachment region (SAR) and β-globin matrix attachment region (MAR) to promote the position-independent expression of a recombinant gene and strong promoter of mouse EF1 α modified with the TATA box of CHO EF1 α promoter. In addition, it has an efficient polyadenylation signal and transcription terminator. Modification of the translation initiation site of rhActivin A with the Kozak sequence was performed during the DNA cloning process.[24] Following the adaptation in EX-CELL® CD CHO medium (Merck) supplemented with 8 mM L-glutamine and HT, DHFR-deficient CHO cells (CHO DG44) were used as host cells for the transfections. Transfection of the host cells with rhActivin A expression plasmid DNA (5 µg), along with the pDCH1P plasmid (28 ng) containing the dhfr gene as a selection marker, was performed by electroporation as previously described.[25] The pDCH1P plasmid is 5614 bp DNA and able to express dhfr with the structural gene of CHO dhfr and its original promoter.
Serum microRNA profiles among dioxin exposed veterans with monoclonal gammopathy of undetermined significance
Published in Journal of Toxicology and Environmental Health, Part A, 2020
Weixin Wang, Youn K. Shim, Joel E. Michalek, Emily Barber, Layla M. Saleh, Byeong Yeob Choi, Chen-Pin Wang, Norma Ketchum, Rene Costello, Gerald E. Marti, Robert F. Vogt, Ola Landgren, Katherine R. Calvo
Quantitative real-time PCR (qRT-PCR) was used for the quantitation of miRNAs. qRT-PCR was performed with a polyadenylation step prior to reverse transcription as described previously (Luo et al. 2012) with some modifications. Briefly, 7 µl RNA was poly-adenylated using the poly-(A) polymerase (New England Biolabs, Ipswich, MA) according to the manufacturer’s recommendations. Reverse transcription was conducted utilizing an anchor primer (Supplementary Table 1). Real-time PCR was performed on the StepOnePlus instrument (Life Technologies, Grand Island, NY) using a forward primer containing mature microRNA sequence and a universal reverse primer and a FAM-ZEN-IABK-labeled TaqMan probe (Integrated DNA Technologies, Coralville, IA) (Supplementary Table 1). Cycle threshold (Ct) values for each miRNA were employed to determine miRNA levels and normalized to cel-miR-39 level, which were calculated as 2−ΔCt (Livak and Schmittgen 2001).