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Mechanisms of Fibril Formation and Cellular Response
Published in Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin, XIth International Symposium on Amyloidosis, 2007
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin
products were cloned with the TA Cloning Kit (Invitrogen) and two clones with expected insertion size from each PCR product were subjected to DNA sequencing forwards and backwards. Five clones yielded the same k1 O18/O8 sequences, with which the tryptic fragment sequences from the MS/MS experiments aligned with full identity. Genomic DNA was extracted from the same tissue for PCR and subsequent DNA sequencing experiments on apoE exons, and showed that the patient was homozygous for the apoE3 gene. ApoE3 protein and its fragments may bind to the periphery of amyloid fibrils. Immunogold particles conjugated to the second antibody were aligned along amyloid fibrils by transmission electron microscopy when the primary antibody was specific to apoE, and the extracted amyloid fibrils were able to absorb externally applied apoE protein.
Ionizing radiation does not impair the mechanisms controlling genetic stability during T cell receptor gene rearrangement in mice
Published in International Journal of Radiation Biology, 2018
Serge M. Candéias, Sylwia Kabacik, Ann-Karin Olsen, Dag M. Eide, Dag A. Brede, Simon Bouffler, Christophe Badie
20 μl of the second PCR reaction was separated on 1.5% agarose gel and appropriate bands were cut and extracted with Zymoclean™ Gel DNA Recovery Kit (Zymo Research) according to manufacturer’s protocol. The purified PCR products were cloned using TOPO®TA Cloning®Kit for Sequencing (Life Technologies) according to manufacturer’s protocol. Five to 10 colonies for each PCR product were picked and used for setting up a bacterial culture. From each culture, plasmids were extracted using Zyppy™ Plasmid Miniprep Kit (Zymo Research). Around 10 ng of purified plasmid were used for PCR amplification of the insert using AccuStart™ II Taq DNA Polymerase (Quanta Bioscience) and 1.5 mM magnesium chloride, 200 μM each dNTP and 400 nM M13-Forward and M13-Reverse primers in 20 μl reaction. The cycling conditions were as follows: 3 min at 94 °C then 35 cycles of 10 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C, with 10 min final extension. The PCR reaction was separated on 1.5% agarose gel and appropriate bands were cut and extracted with Zymoclean™ Gel DNA Recovery Kit (Zymo Research) according to manufacturer’s protocol. The purified PCR products were sent for sequencing to Source BioSicence (Source BioSicence, Oxford, UK) at a concentration of 1 ng/µl per 100 bp. The T3 and T7F primers were used for sequencing.
Transcriptional control of the MUC16 promoter facilitates follicle-stimulating hormone peptide-conjugated shRNA nanoparticle-mediated inhibition of ovarian carcinoma in vivo
Published in Drug Delivery, 2018
Ming-Xing Zhang, Shan-Shan Hong, Qing-Qing Cai, Meng Zhang, Jun Chen, Xiao-Yan Zhang, Cong-Jian Xu
The predicted promoter sequences were generated from oligonucleotides by PCR. The PCR products were cloned into TA cloning vector pMD18-T (TAKARA Bio Inc., Japan), and the vector was transfected into DH5a competent cells. Then, the TA cloning vector was digested and ligated with the basic vector pGL4.10[luc2] (Promega Corporation, USA) at the XhoI and BglII restriction sites. The pGL4.10 vector had no promoter and encodes the luciferase reporter gene luc2. The ligated product was named pGL-MUC16. The CMV promoter was also ligated to pGL4.10, and the product was named pGL-CMV and used as the positive control in the experiments described below.
Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant biotherapeutic antibodies from CHO cells
Published in mAbs, 2023
Emma Kelsall, Claire Harris, Titash Sen, Diane Hatton, Sarah Dunn, Suzanne Gibson
Sequence analysis of individual HC transcripts from CHO pools was performed by isolation of RNA using an RNAeasy mini kit (Qiagen, Cat# 74104), followed by RT-PCR of the HC mRNA using a Transcriptor one-step RT-PCR kit (Roche, Cat# 04655877001) and primers spanning the CH1- hinge-CH2 region. The resulting individual cDNA fragments were cloned into a plasmid using a TOPO-TA cloning plasmid kit and used to transform E. coli, according to manufacturer’s instructions (Invitrogen, Cat# 450030). The resulting bacterial colonies were analyzed using Sanger sequencing.