Tissue-specific Knockout
Tissue-specific Knockout - Related Videos
03:58
Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System
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Cited by 6 •
2023
In this protocol, we describe the technical procedures to generate brown adipose tissue (BAT)-specific knockout mice leveraging a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The described steps include the design of the sgRNAs, the preparation of the AAV-sgRNA particles, and the microinjection of AAV into the BAT lobes.
05:25
Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement
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Cited by 2 •
2023
This study provides a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describes methods for analyzing alveolar bone remodeling during orthodontic tooth movement, thus shedding light on skeletal mechanical biology.
09:42
Generating Transgenics and Knockouts in Strongyloides Species by Microinjection
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Cited by 13 •
2021
The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S. stercoralis and S. ratti.
11:49
Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System
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2023
This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.
08:34
Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink
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Cited by 59 •
2016
We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.
FAQs
The key restriction comes from where Cre recombinase is produced. A tissue-specific promoter drives Cre expression in the selected tissue or cell type, so Cre recognizes the two loxP sites and excises the DNA between them in those cells. This links promoter activity to the location of gene disruption, rather than applying the change across the entire organism.
The loxP sites act as positional boundaries for recombination. When Cre is active, it targets those paired sequences and removes the intervening segment, disabling the gene in the affected cells. Without this arrangement, tissue-restricted Cre expression would not provide the same defined genetic switch for the selected target. Thus, loxP placement is central to conditional control.
A whole-body knockout can combine effects from many tissues, making it difficult to identify where a gene normally acts. Restricting gene loss to one tissue preserves the gene's function elsewhere, helping researchers attribute observed biological changes to the selected tissue or cell type. This separation supports clearer analysis of physiology, development, and disease mechanisms.
In cells where the tissue-specific promoter does not activate Cre, the loxP-flanked gene is not excised under the described strategy. Its function therefore remains available outside the selected tissue or cell type. This preserved activity creates an important internal contrast between cells with tissue-restricted gene disruption and those retaining the target gene.
The target gene is first arranged with loxP sequences flanking the DNA region intended for excision. A Cre recombinase gene is then placed under the control of a promoter active in the tissue or cell type of interest. In cells where that promoter functions, Cre removes the intervening target sequence, producing the intended localized gene disruption.
This approach can reveal whether a gene's role differs among tissues or cell types without eliminating its function throughout the organism. Researchers can use that distinction to investigate tissue-specific contributions to development, physiology, and disease mechanisms. The resulting evidence can also clarify whether a gene or its tissue-specific activity represents a potential therapeutic target.