Episomal Gene Targeting
Episomal Gene Targeting - Related Videos
08:56
Recombineering and Gene Targeting
0 Views •
2023
One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that allow them to be joined together. However, this technique has certain restrictions that limit its applicability for large or complex DNA construct generation. A newer technique that addresses some of these shortcomings is recombineering, which modifies DNA using homologous recombination (HR), the exchange between different DNA molecules based...
09:56
Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins
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2025
This protocol describes a non-integrative, episomal CRISPR/dCas9-based system for targeted epigenetic editing in K562 cells, combining dCas9-DNMT3A and dCas9-HDAC1 effectors with specific sgRNAs to induce locus-specific DNA methylation with precision and reduced off-target effects.
10:52
Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids
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Cited by 26 •
2015
Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.
08:48
Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast
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Cited by 5 •
2017
A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.
05:33
Biolistic-Mediated Gene Transfer: A Technique to Deliver Gene of Interest in Target Cells via a Biolistic Gene Gun
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2025
In this video, we demonstrate the protocol of biolistic gene gun-mediated gene delivery in cultured cells. This method provides a simple method of stable genetic transformation using a minimal quantity of DNA.
FAQs
Targeting specificity comes from two cooperating elements: sequence homology in a donor template or recognition by a programmable nuclease. These features direct repair toward the chosen genomic locus, whereas the episomal carrier supplies the relevant sequence or guide without becoming part of the cell’s permanent DNA. This separation allows locus-directed modification while the carrier can later be diluted or removed.
Each component supports a different stage of the strategy. A guide helps direct a programmable nuclease to the chosen locus, while a donor template provides sequence information for homology-based repair. A selectable marker can support identification of cells receiving the vector. Together, these elements connect vector entry, locus recognition, sequence modification, and marker expression.
Here, the targeting vector itself is not required to integrate into the genome. The desired locus can still be modified or regulated through guide-directed or homology-based repair, while the carrier remains extrachromosomal and may be diluted or removed. This is useful when researchers need gene perturbation without maintaining a permanently integrated delivery construct.
An episomal molecule is introduced into cells with the sequence needed for the experiment, such as a guide, donor template, or selectable marker. After entry, the episome expresses these elements, and homology or a programmable nuclease directs repair at the selected locus. Researchers can then examine the resulting gene modification or regulation after the carrier is diluted or removed.
It enables functional analysis of host and pathogen genes without making persistence of the targeting vector the central experimental feature. In this setting, investigators can examine genes that influence immune recognition, pathogen replication, or virulence. The approach therefore links a defined genetic perturbation to biological questions relevant to host defense and infection while retaining controlled delivery.
Dilution or removal of the episomal carrier separates the intended locus-directed change from continued presence of the targeting vector. Researchers can therefore assess consequences of modifying or regulating the chosen genomic site under conditions where the episomal construct is no longer maintained. This feature matters when permanent vector integration is undesirable for controlled gene perturbation.