Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5755190 | PMC |
| http://dx.doi.org/10.3791/56319 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Systematic deletion of symmetrical exons reveals new therapeutic targets for exon skipping antisense oligonucleotides.
NAR Mol Med
October 2024
Center for Genetic Diseases, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Rd, North Chicago, IL 60064, USA.
Article Synopsis
- There is a significant need for new treatments targeting diseases caused by premature termination codons (PTCs), which lead to faulty proteins.
- Splice-switching antisense oligonucleotides (ASOs) can help by inducing exon skipping, effectively removing PTCs from mRNA and potentially restoring protein function if the remaining exons are in the correct reading frame.
- The research focuses on the cystic fibrosis transmembrane regulator (CFTR) gene, demonstrating that ASOs can restore CFTR function in airway cells from individuals with PTC-causing mutations, showing the potential for ASO therapies across similar multi-exon genes.
Development of a baculoviral CRISPR/Cas9 vector system for beta-2-microglobulin knockout in human pluripotent stem cells.
Mol Genet Genomics
August 2024
Department of Epidemiology and Health Statistics, School of Public Health, Guangdong Medical University, Dongguan, Guangdong, China.
Derivation of hypoimmunogenic human cells from genetically manipulated pluripotent stem cells holds great promise for future transplantation medicine and adoptive immunotherapy. Disruption of beta-2-microglobulin (B2M) in pluripotent stem cells followed by differentiation into specialized cell types is a promising approach to derive hypoimmunogenic cells. Given the attractive features of CRISPR/Cas9-based gene editing tool and baculoviral delivery system, baculovirus can deliver CRISPR/Cas9 components for site-specific gene editing of B2M.
View Article and Find Full Text PDFHIF3A gene disruption causes abnormal alveoli structure and early neonatal death.
PLoS One
May 2024
Center for Integrated Medical Education and Regional Symbiosis, Asahikawa Medical University, Asahikawa, Japan.
Transcriptional response to changes in oxygen concentration is mainly controlled by hypoxia-inducible transcription factors (HIFs). Besides regulation of hypoxia-responsible gene expression, HIF-3α has recently been shown to be involved in lung development and in the metabolic process of fat tissue. However, the precise mechanism for such properties of HIF-3α is still largely unknown.
View Article and Find Full Text PDFInduction of Oxidative Stress in Sirtuin Gene-Disrupted Ashbya gossypii Mutants Overproducing Riboflavin.
Mol Biotechnol
May 2024
Molecular and Biological Function Research Core, Research Institute of Green Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, Japan.
AgHST1 and AgHST3 genes encode sirtuins that are NAD-dependent protein deacetylases. According to previous reports, their disruption leads to the overproduction of riboflavin in Ashbya gossypii. In this study, we investigated the potential causes of riboflavin overproduction in the AgHST1Δ and AgHST3Δ mutant strains of A.
View Article and Find Full Text PDFInhibition of Viral Replication Reduces Transcriptionally Active Distinct Hepatitis B Virus Integrations With Implications on Host Gene Dysregulation.
Gastroenterology
April 2022
Gilead Sciences Inc., Foster City, California, USA. Electronic address:
Background & Aims: Hepatocellular carcinogenesis of hepatitis B virus (HBV) infection may arise from integration of viral DNA into the host genome. We aimed to gauge the effect of viral inhibition on transcriptionally active HBV-host integration events and explore the correlation of viral integrations with host gene dysregulation.
Methods: We leveraged data and biospecimens from an interventional trial, in which patients with HBV viremia above 2000 IU/mL and minimally raised serum liver enzyme were randomized to receive tenofovir disoproxil fumarate (TDF) or placebo for 3 years.
Want AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!