Targeting Vector Construction
Targeting Vector Construction - Related Videos
09:02
Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Cited by 4 •
2015
Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.
09:16
A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors
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Cited by 13 •
2016
Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.
16:04
A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells
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Cited by 6 •
2013
Here, we use retroviral transduction and concatemeric transfection to create a cell line that can express the components of a lentiviral vector (LV) in the absence of tetracycline. This LV encodes GFP and is pseudotyped with a glycoprotein, SVGmu, which is specific for a receptor on dendritic cells.
12:59
High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots
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Cited by 37 •
2016
Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.
07:00
Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants
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Cited by 23 •
2010
In this article, we show a method to make glass capillary needles with a 50-μm lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.
FAQs
Homologous sequences match the corresponding target region on a chromosome, allowing the vector and genomic DNA to align during homology-directed recombination. This alignment is the central targeting step because it helps place a planned deletion, reporter gene, or other modification at the intended locus rather than treating the vector as unrelated DNA.
The vector’s components serve different design purposes. Homologous regions specify where alignment can occur, while the planned modification determines the genetic change to be introduced. Selectable elements are included as part of the construct to support recovery of cells carrying the vector-based change. Together, these components connect locus choice, genetic outcome, and identification of modified cells.
Knockouts, knock-ins, and conditional alleles address different developmental questions. A knockout can remove gene function, a knock-in can add a reporter or another planned sequence, and a conditional allele can restrict when or where the genetic change is examined. Selecting among them affects how directly researchers can connect gene activity with patterning, differentiation, or tissue formation.
A logical workflow begins by selecting the genomic locus, designing homologous sequences around the target region, and placing the intended modification and selectable elements within the vector. The construct is then delivered into cells, where homology-directed recombination can align it with the chromosome and integrate the planned change. Each stage links vector architecture to the resulting genetic model.
Developmental biologists use these constructs when they need to test how a particular gene contributes to embryonic patterning, cell differentiation, or tissue formation. The approach is most informative when a defined genomic change can be connected to a resulting developmental phenotype. Reporter-containing designs can investigate gene-related patterns, whereas other designs alter gene function directly.
The resulting genetic models can connect a targeted locus to observable developmental or disease-related phenotypes. Comparing a gene knockout, a knock-in, or a conditional allele can help distinguish effects of gene loss, an added sequence, or a context-dependent genetic change. This supports interpretation of how specific genes contribute to development and disease-related traits.