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Protocol for conditional mutagenesis in zebrafish germ cells using Tol2 transposon and a CRISPR-Cas9-based plasmid system.
STAR Protoc
December 2024
College of Life Sciences, Northwest Normal University, Lanzhou 730070, China. Electronic address:
Here, we present a protocol for conditional mutagenesis in zebrafish germ cells using Tol2 transposon and a CRISPR-Cas9-based plasmid system. We describe steps for conditional mutagenesis plasmid construction, zebrafish embryo microinjection, and screening for green fluorescence in the heart. This protocol is simple to execute, time efficient, and multifunctional, enabling the disruption of genes in zebrafish germ cells to be conducted with ease.
View Article and Find Full Text PDFIn Vivo Selection of S/MAR Sequences to Favour AAV Episomal Maintenance in Dividing Cells.
Int J Mol Sci
November 2024
Vivet Therapeutics S.L., 31008 Pamplona, Spain.
Adeno-associated viral (AAV) vector-mediated gene therapy has emerged as a promising alternative to liver transplantation for monogenic metabolic hepatic diseases. AAVs are non-integrative vectors that are maintained primarily as episomes in quiescent cells like adult hepatocytes. This quality, while advantageous from a safety perspective due to a decreased risk of insertional mutagenesis, becomes a disadvantage when treating dividing cells, as it inevitably leads to the loss of the therapeutic genome.
View Article and Find Full Text PDFAlkA Glycosylase and AlkB Dioxygenase Constitute an Effective Protective System for Endogenously Arising Acrolein: E. coli AlkA Glycosylase Excises Acrolein Adduct to Adenine.
J Mol Biol
December 2024
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland. Electronic address:
Acrolein (ACR) is a ubiquitous environmental pollutant but also formed endogenously as a metabolite in oxidative stress conditions. Its adduct to adenine 1,N-α-hydroxypropanoadenine (HPA) is a mutagenic lesion effectively repaired by the AlkB dioxygenase. Here, we provide in vivo, in vitro, and in silico evidence that it is also the substrate for the AlkA glycosylase.
View Article and Find Full Text PDF5'-untranslated region sequences enhance plasmid-based protein production in .
Front Microbiol
November 2024
Molecular Enzyme Technology and Biochemistry (MEB), Environmental Microbiology and Biotechnology (EMB), Centre for Water and Environmental Research (CWE), Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany.
, a thermoacidophilic archaeon of the phylum Thermoproteota (former Crenarchaeota), is a widely used model organism for gene deletion studies and recombinant protein production. Previous research has demonstrated the efficacy of the promoter (P), providing low basal activity and high pentose-dependent induction. However, the available expression vector does not include a 5'-terminal untranslated region (5'-UTR), a typical element found in bacterial expression vectors that usually enhances protein production in bacteria.
View Article and Find Full Text PDFCell-Specific Control of Mammalian Gene Expression Using DNA Repair Inducible Ribozyme Switches.
Angew Chem Int Ed Engl
December 2024
State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biomedical Sciences, Hunan University, Changsha, 410082, China.
The ability to control gene expression is vital for elucidating gene functions and developing next-generation therapeutics. Current techniques are challenged by the lack of cell-specific control designs or immunogenicity risk from foreign proteins. We develop a DNA repair inducible ribozyme switch that enables cell-specific control of gene expression in cells and in vivo.
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