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Investigating the Role of Two-Pore Channel 2 (TPC2) in Zebrafish Neuromuscular Development
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sarah E. Webb, Jeffrey J. Kelu, Andrew L. Miller
To construct the tpcn2 gRNA, a linear DNA template was first amplified from the gRNA scaffold vector via PCR (Kelu et al., 2017), using short oligonucleotides as primers. The forward primer contained the T7 promoter (for in vitro transcription), followed by the 20-bp tpcn2 gRNA target sequence (excluding the PAM) and then the 5’ portion of the gRNA scaffold, whereas the reverse primer contained only the 3’ portion of the gRNA scaffold. The tpcn2 gRNA was then synthesized via in vitro transcription and purified using phenol/chloroform extraction. Similarly, the Cas9 mRNA was synthesized via in vitro transcription using the linearized DNA template obtained from the pT3TS plasmid vector (Jao et al., 2013), and then purified. To perform gene-editing, a mixture of the tpcn2 gRNA and Cas9 mRNA was co-injected into the blastodisc of wild-type zebrafish embryos at the 1-cell stage (Figure 5.2Bi; Kelu et al., 2017). To evaluate the mutation efficiency in the injected embryos (F0), the restriction endonuclease digestion assay was performed. Several injected embryos were sacrificed at ~24 hpf, and the genomic DNA was extracted. The target region was then amplified via PCR using a primer pair flanking tpcn2exon 9 (Figure 5.2A), after which the amplicons were restriction-digested using AvaI and separated via agarose gel electrophoresis. Finding the ratio of the intensity of the undigested band and the two digested bands on the gel indicates the proportion of mutated versus wild-type tpcn2 alleles in the injected embryos. The tpcn2 mutations were further characterized using TOPO cloning followed by Sanger sequencing analysis, and were shown to contain both insertions and deletions from within the tpcn2 locus (Figure 5.2C; Kelu et al., 2017).
Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Hyun Myung Lee, Rudolf Andrys, Jakub Jonczyk, Kyuneun Kim, Avinash G. Vishakantegowda, David Malinak, Adam Skarka, Monika Schmidt, Michaela Vaskova, Kamil Latka, Marek Bajda, Young-Sik Jung, Barbara Malawska, Kamil Musilek
The hrAChE and hrBChE were prepared as recombinant proteins at the University of Hradec Kralove. For their production, the mammalian expression system was used. Briefly, the DNA sequence encoding human AChE and BChE was obtained from UniProtKB Server (www.uniprot.org, accession numbers: P22303 and P06276) and de novo synthesised as GeneArt Strings DNA fragments by GeneArt Gene Synthesis Service (Thermo Fisher Scientific, Waltham, MA). The DNA fragments were PCR-amplified using gene-specific primers, adding the DNA sequence for C-terminal 6 × His-tag. The amplicons were inserted into the mammalian pcDNA3.4 vector by TOPO cloning technology, and the final DNA constructs were verified by Sanger sequencing (ABI PRISM 3130xl). For protein expression, the DNA constructs were transiently transfected into Hek293 derivatives. Recombinant proteins were collected from culture supernatant six days later and stored at −80 °C for further purification.
Maintenance of the aseptic working field during endodontic treatment
Published in Acta Odontologica Scandinavica, 2019
Line Rørslett Hardersen, Morten Enersen, Anne Karin Kristoffersen, Dag Ørstavik, Pia Titterud Sunde
Any PCR product from the samples was visualized with gel electrophoresis. The products were ligated into a TOPO cloning vector and transformed into E.coli (Topo® TA Cloning® kit K4500-01, Invitrogen). Twenty colonies of each sample were analysed. PCR with M13 primers in front of the insert and the M13 PCR product were sequenced using BigDye® Terminator v1.1 cycle Sequencing Kit and ABI 3730 sequencer (Applied Biosystems). All DNA sequences were analysed, trimmed, and aligned using FinchTV software (version 1.5.0; https://digitalworldbiology.com/FinchTV). The partial gene sequences were identified by a BLAST analysis with 16S rRNA GeneBank database search on the Human Oral Microbiome database (HOMD) [17].
ABO antigen levels on platelets of normal and variant ABO blood group individuals
Published in Platelets, 2019
Xianguo Xu, Fang Xu, Yanling Ying, Xiaozhen Hong, Ying Liu, Shu Chen, Ji He, Faming Zhu, Wei Hu
If novel nucleotide mutations or ambiguous genotypes of ABO or FUT1 loci were found, the haplotype analysis was performed using a TOPO cloning kit (Invitrogen, Carlsbad, CA, USA) as previously described [18]. All primers for PCR amplification and DNA sequencing are listed in Table I. Alleles were named according to International Society of Blood Transfusion [19].