Conditional Knockout Vectors
Conditional Knockout Vectors - Related Videos
10:42
A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
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Cited by 21 •
2017
We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.
08:22
Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference
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Cited by 2 •
2013
This article describes how to microinject viral vectors into mouse brain and then test in a conditioned place preference paradigm that includes an acquisition, extinction and reinstatement phase.
08:39
Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord
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Cited by 18 •
2013
Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.
04:13
CRISPR Concatemer-Mediated Multiple Gene Knockout: A Technique to Simultaneously Knockout Multiple Genes by Non-Homologous End-Joining Pathway in Mouse Intestinal Cells
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2025
This video describes a gene knockout technique using a CRISPR-concatemer to simultaneously knock out multiple genes in cultured mouse intestinal organoid cells. This method is used to knock out a diseased gene and to elucidate the function of a gene and its paralogues.
11:53
A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer
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Cited by 17 •
2017
This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.
FAQs
An essential segment of the target gene is flanked by loxP sites, producing a floxed allele. When Cre recombinase is expressed, it recognizes these sites and excises the intervening DNA. Gene disruption therefore depends on where and when Cre is present, allowing deletion to occur in defined biological settings rather than throughout the organism.
The promoter controlling Cre expression is the main determinant of spatial specificity. A tissue-specific promoter restricts recombinase production to selected cell populations, so the floxed gene is excised primarily there. This arrangement allows the same target allele to produce different experimental outcomes depending on which promoter drives Cre expression.
A tissue-specific system limits Cre expression according to the promoter’s activity in particular cells or tissues. An inducible system adds control over timing, enabling recombinase expression at a defined stage rather than throughout the entire relevant period. This distinction is important when researchers need to separate gene function in development from gene function later in life.
Conditional deletion is designed to remove an essential segment of the target gene, increasing the likelihood that Cre-mediated excision will disrupt gene function. Compared with a conventional organism-wide knockout, this strategy can reveal effects in a chosen tissue or time window while avoiding changes that might arise from complete deletion across the entire organism.
Researchers first arrange loxP sites around an essential segment of the gene, creating the floxed allele. They then use Cre expression controlled by a tissue-specific or inducible promoter. The resulting system is evaluated in the selected cells, tissues, or time period to determine how removing that gene segment changes biological function.
They are particularly useful for studying developmental processes, disease mechanisms, and functions that differ among tissues. Restricting gene disruption can help researchers examine a target in the relevant biological context without the confounding effects of an organism-wide deletion. This makes the approach valuable for linking gene activity to specific physiological or pathological outcomes.