Genetic Approaches to Physiological Problems II Notes
Genetic Approaches to Physiological Problems II
Introduction
Presenter: Annette de Kloet, Ph.D.
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Outline
Application of genetic approaches
Conditional gene manipulations using the Cre/lox system
Cre recombinase
LoxP sites
Cell type-specific gene deletion
Cell type-specific gene ‘knock-in’
Tamoxifen- or tetracycline-inducible Cre recombinase
Reporter mouse strains
Genetic Modifications to the Mouse Genome
Various techniques to modify the genome of mice including:
Loss of function mutations
Example: knockout mice
Gain of function mutations
Example: overexpression of genes of interest, expression of reporter genes
Conditional Gene Manipulation
Using the Cre/lox system
Conditional Gene Manipulation: Cre/LoxP System
Cre recombinase
An enzyme derived from the P1 bacteriophage.
Catalyzes site-specific recombination of DNA between two loxP sites.
LoxP Sites
LoxP sites are small sequences located in non-coding regions.
Ensures they do not interfere with the expression of gene X in cells not expressing Cre.
Each loxP site is a 34 base pairs (bp) sequence, targeted by Cre recombinase.
Generating Dual LoxP Knock-In Mice Using CRISPR/Cas9
Targeting exons in the mouse genome through the following methods:
Microinjection of a cocktail that includes:
guide RNAs (gRNAs)
loxP sequences
Single-Stranded Oligonucleotide Donors (SSODN)
NHEJ (Non-Homologous End Joining) and Homology-Directed Repair processes involved.
Result: Floxed targeting exons integrated into the mouse genome.
Application of Cre/LoxP System
Specific deletion of gene X in liver cells for evaluating its physiological and pathophysiological role.
Cell-Type Specific Gene Deletion Using Cre/LoxP System
Example of the liver-specific Cre transgene:
B6.Cg-Tg(Alb-Cre) 21Mgn/J (Stock No. 003574)
Mechanism:
The allele containing loxP sites flanking gene X undergoes recombination when expressed in liver cells due to the presence of Cre recombinase.
Breeding Strategy for Cre/LoxP System
Breeding for a cohort of mice with specific genetic modifications, including control mice.
Ensure to counterbalance with littermates to control for litter effects.
Littermate matching reduces variability due to background strain effects from mixed strains.
Controls and Considerations
Include various control groups:
Mice with neither loxP nor Cre.
Mice expressing only Cre.
Mice with loxP-flanked genes only.
Importance of controlling for environmental effects and genetic backgrounds, particularly if utilizing mixed strains.
Other Applications of the Cre/Lox System
Cell-type specific gene expression through ‘knock-in’ strategy.
Example: Expression of Gene X only in specific cell types containing Promoter Z.
Usage of ROSA26 locus for ubiquitous gene expression.
Disadvantages of Conditional Knock-out/Knock-in Approaches
Not all tissue-specific promoters maintain specificity.
High costs and duration of experimentation.
Early gene deletion may create undesired developmental effects.
Solutions to Address Disadvantages
Solution 1: Inducible conditional gene targeting
Inducible Cre (Tamoxifen or Tetracycline-inducible)
Cre-recombinase fused to a modified Estrogen Receptor (ER) controlled by tissue-specific promoter.
In the absence of Tamoxifen, the Cre-ER fusion is kept in the cytosol by heat shock protein 90.
Upon Tamoxifen exposure, the ER translocates to the nucleus, where Cre performs recombination and creates a knockout.
Note: Alternative inducible systems exist (e.g., tetracycline-induced).
Solution 2: Virally-mediated gene transfer of cre-recombinase
Utilizes Adeno-associated viruses to express Cre in neurons (e.g., AAV-Cre).
Reporter Mouse Strains: Rationale for Development
Useful when the gene of interest can’t be easily identified with existing methods like immunohistochemistry.
Enables visualization of specific cell types without postmortem processing, facilitating techniques such as patch-clamp electrophysiology.
Examples of Reporter Mouse Strains
Example 1: Using the Cre/lox system
Ubiquitous promoter linked with STOP sequence preceding a GFP (Green Fluorescent Protein) that is activated only when Cre is present.
Example 2: Transgenic Reporter Mouse
Enhanced green fluorescent protein (EGFP) was inserted into the Agtr2 BAC clone at the start codon of the first coding exon.
This construct results in EGFP expression governed by all regulatory sequences of the Agtr2 BAC gene.
Example 3: Knock-in of Reporter Gene
Reporter gene expressed after the gene of interest using gene targeting methods (homologous recombination) or gene editing techniques (CRISPR/Cas9).
Strengths and Weaknesses of Reporter Approaches
CRE/LOX SYSTEM
Strength: Flexible and permits numerous applications.
Weakness: Potential for false positives.
GENE TARGETING
Strength: Endogenous expression levels are often similar.
Weakness: Limited application possibilities, resulting in reduced utility.
Summary of Key Concepts
Distinction between transgenic approaches and gene targeting.
Overview of CRISPR/Cas9 gene editing technology.
Importance and methodology of conditional gene manipulations using the Cre/lox system.
Functionality and utility of various reporter mouse strains.