Genetic Approaches to Physiological Problems II Study Notes

Genetic Approaches to Physiological Problems II

Overview

  • This lecture focuses on genetic approaches to address physiological problems, presented by Annette de Kloet, Ph.D.

  • Importance of the content is noted along with copyright protection against unauthorized distribution.

Outline of the Lecture

  • 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

  • Loss of function mutations

    • Example: Knock-out mice

  • Gain of function mutations

    • Example: Overexpression of genes of interest, expression of reporter genes

  • Conditional Gene Manipulation

    • Utilization of 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

    • Comprised of a 34 base pair (bp) sequence that serves as the target for Cre recombinase.

    • It is situated in non-coding regions to avoid interference with gene X expression in cells that do not express Cre.

Using CRISPR/Cas9 to Generate Dual loxP Knock-in Mouse

  • Mouse genome targeting involving various elements:

    • Targeting exons

    • Microinjection

    • gRNAs (guide RNAs)

    • LoxP sequences

    • Single Strand Oligonucleotides (SSODN)

    • Employing Non-Homologous End Joining (NHEJ) + Homology-directed Repair (HDR)

Example: Deleting Gene X

  • Application of the Cre/lox system to specifically delete gene X from liver cells to evaluate its role in various physiological and pathophysiological contexts.

Cell-type Specific Gene Deletion Using Cre/LoxP System

  • Description of the process utilizing Alb (albumin) gene which drives liver-specific Cre transgene

    • Example: B6.Cg-Tg(Alb-Cre) 21Mgn/J (Stock No. 003574)

    • Points out the breeding of conditional knockout mice with targeted deletions in specific tissues.

Diagram Representations

  • Cre-LoxP Gene Targeting:

    • Indicates how Gene X is 'floxed' using loxP sites to allow specific knockout.

Appropriate Controls and Considerations

  • Conditional (cell-type-specific) knockouts require careful control:

    • Include mice that do not contain the loxP allele or the Cre

    • Mice expressing only Cre-recombinase

    • Mice expressing only the loxP-flanked gene

  • Importance of counterbalancing groups based on littermates to control for litter effects, particularly with mixed background strains.

Background Information

  • The Jackson Laboratories maintains background strain information for over 4,000 different mouse strains, illustrating genetic variability concerns in experiments.

  • Notable strains include the C57BL6/J for diet-induced obesity studies and BPH/2J for hypertension.

Breeding Considerations

  • Detailed explanation of a breeding strategy for cohort generation with Flox Mice as controls:

    • Example: Crossing configurations for Cre+/− and GeneXloxp/loxp mice to create genetic variability.

  • Stress the importance of litter effects control especially under mixed background strains.

Other Applications of the Cre/lox System

  • Beyond gene deletion, the system can be used for cell-type specific gene expression or 'knock-in' approaches involving target genes.

    • Usage of Promoter Z to control expression in certain cell types.

Challenges of Conditional Knock-Out/Knock-In Approaches

  • Specificity of tissue-specific promoters can vary; some may not be truly specific to the target tissue.

  • Developmental timing and costs associated with these methods can present limitations.

    • Early deletion of the gene of interest during development can lead to unintended effects.

Solutions for Inducible Conditional Gene Targeting

  • Solution 1: Inducible Cre

    • Fusing Cre-recombinase with a modified Estrogen Receptor (ER) controlled by a tissue-specific promoter.

    • In its basal state, the Hsp90 protein retains the ER in the cytosol.

    • Upon tamoxifen exposure, the ER is released, allowing Cre translocation to the nucleus for gene recombination.

    • Note: Other systems, such as tetracycline-inducible methods, exist.

  • Solution 2: Virally-mediated Gene Transfer

    • Utilizes Adeno-associated virus to infect neurons to express Cre.

Reporter Mouse Strains: Development Rationale

  • Reporter mice are developed for genes that are difficult to identify through traditional approaches, such as immunohistochemistry.

  • Offers visualization of specific cell types without the need for extensive postmortem processing, valuable for procedures like patch-clamp electrophysiology.

Examples of Reporter Mouse Strains

  • Example 1: Cre/lox system with a ubiquitous promoter driving expression of a gene like GFP in certain cells.

  • Example 2: Transgenic mice with an enhanced green fluorescent protein (EGFP) inserted into the Agtr2 BAC clone to visualize expression regulated by Agtr2's sequences.

  • Example 3: Implementing homologous recombination or CRISPR/Cas9 for the expression of a reporter gene adjacent to the endogenous gene.

Strengths and Weaknesses of the Approaches for Generating Reporter Mice

  • CRE/LOX SYSTEM

    • Spatial expression: Potential for false positives

  • GENE TARGETING

    • Analogous to endogenous levels of expression, less risk of false positives

  • GENOMIC FRAGMENT

    • Flexibility and more utility in various applications

  • TRANSGENE

    • Limited to specific applications, generally lacks broader versatility.

Summary of Key Concepts

  • Differentiation between Transgenic versus gene targeting approaches

  • Overview of CRISPR/Cas9 gene editing technology

  • In-depth look at conditional gene manipulations using the Cre/lox system

  • Analysis of reporter mouse strains and their applications in genetic research.

Genetic Approaches to Physiological Problems II
Overview

  • This lecture focuses on genetic approaches to address physiological problems, presented by Annette de Kloet, Ph.D.

  • Importance of the content is noted along with copyright protection against unauthorized distribution.

Outline of the Lecture - Key Concepts for Memorization

To memorize the lecture's scope, focus on these main application areas:

  1. Application of genetic approaches: Understand the "why" – using genetics to study physiology.

  2. Conditional gene manipulations using the Cre/lox system: This is a central technique. Remember its components and variations:

    • Cre recombinase: The enzyme.

    • LoxP sites: The target sequences.

    • Cell type-specific deletion/knock-in: The main uses.

    • Inducible Cre: Tamoxifen/tetracycline for timed control.

    • Reporter mouse strains: For visualization.

  3. Genetic Modifications to the Mouse Genome: Types of changes you can make.

  4. CRISPR/Cas9: How loxP sites are inserted.

  5. Reporter Mouse Strains: Their development, examples, and evaluation (strengths/weaknesses).

  6. Controls & Breeding: Essential experimental design aspects.

  7. Challenges & Solutions: Common pitfalls and how to overcome them.

Genetic Modifications to the Mouse Genome

Here's a breakdown of genetic modifications:

Type of Mutation

Description

Example

Loss of Function

Eliminating or reducing the activity of a specific gene.

Knock-out mice

Gain of Function

Enhancing or introducing new gene activity.

Overexpression, Reporter

Conditional Gene Manipulation

Gene modification that can be controlled in specific cells or at specific times.

Cre/lox system

Conditional Gene Manipulation: Cre/LoxP System

Key components for memorization:

Component

Origin

Function

Key Feature / Sequence

Cre recombinase

P1 bacteriophage enzyme

Catalyzes site-specific DNA recombination.

n/a

LoxP sites

34 base pair sequence

Serves as the target for Cre recombinase, flanking the gene of interest.

34 \text{ bp}

Memorization Tip for LoxP: "LoxP" can be remembered as "Location Point" for Cre to act. The "P" in LoxP can also remind you it's a "Pair" (two sites needed).

Using CRISPR/Cas9 to Generate Dual loxP Knock-in Mouse

Elements Involved (Memorization List):

  • Targeting exons: The specific coding regions for modification.

  • Microinjection: Method of delivery into cells.

  • gRNAs (guide RNAs): Direct CRISPR/Cas9 to the target DNA.

  • LoxP sequences: The specific 34 \text{ bp} sites to be inserted.

  • Single Strand Oligonucleotides (SSODN): Provide the repair template.

  • NHEJ (Non-Homologous End Joining) + HDR (Homology-directed Repair): DNA repair mechanisms used.

    • NHEJ: Joins broken DNA ends, often error-prone but can be used for initial breaks.

    • HDR: Uses a template for precise repair (essential for inserting loxP).

Cell-type Specific Gene Deletion Using Cre/LoxP System - The Process (Example: Deleting Gene X in Liver)

Steps for Memorization:

  1. Identify Target Gene: Decide which gene (e.g., Gene X) you want to remove.

  2. 'Flox' Gene X: Engineer mice where Gene X is flanked by LoxP sites. (This makes it "floxed").

  3. Introduce Tissue-Specific Cre: Use a promoter (e.g., Albumin, Alb) to drive Cre recombinase expression ONLY in the desired cell type (e.g., liver).

    • Alb-Cre: Albumin promoter for liver-specific Cre.

  4. Breed: Cross your 'floxed' Gene X mice with the Alb-Cre mice.

  5. Recombination: In liver cells of the offspring, Cre recognizes the LoxP sites and excises Gene X.

  6. Evaluate: Observe the physiological effects of Gene X deletion in the liver.

Appropriate Controls and Considerations for Conditional Knockouts

Key Controls to Remember:

  • Mice without the loxP allele OR without Cre.

  • Mice expressing only Cre-recombinase (no floxed gene).

  • Mice expressing only the loxP-flanked gene (no Cre).

  • Littermate controls: Always balance groups from the same litter to minimize "litter effects" and genetic variability.

Background Strain Importance:

  • C57BL6/J: Common strain for diet-induced obesity studies.

  • BPH/2J: Known for hypertension studies.

  • Memorization Tip: C57BL6/J - "C for Calories" (obesity). BPH/2J - "BP for Blood Pressure" (hypertension).

Challenges of Conditional Knock-Out/Knock-In Approaches & Solutions

Challenge

Explanation

Solution 1: Inducible Cre

Solution 2: Virally-mediated Gene Transfer

Specificity

Tissue-specific promoters may not be truly specific.

Fusing Cre to modified Estrogen Receptor (ER) -> Tamoxifen-inducible activation.

Using Adeno-associated virus (AAV) to infect and express Cre.

Developmental Timing

Early gene deletion can cause unintended developmental issues.

Cre-ER complex retained in cytosol by Hsp90; Tamoxifen releases ER for nuclear translocation.

Delivers Cre directly to specific cells (e.g., neurons) post-development.

Cost / Complexity

Generating and maintaining strains can be expensive and complex.

Offers temporal control, potentially reducing need for multiple breeding cohorts staged differently.

Direct, localized delivery can be more cost-effective for specific applications.

Memorization Tip for Inducible Cre: Think of "TIMER" - Tamoxifen Induces Migration of ER-Recombinase.

Reporter Mouse Strains: Purpose and Examples

Rationale (Why use them?):

  • Difficulty in identifying genes by traditional methods (e.g., immunohistochemistry).

  • Visualization of specific cell types without extensive postmortem processing.

  • Valuable for live cell procedures (e.g., patch-clamp electrophysiology).

Examples (Memorization):

  1. Cre/lox + ubiquitous promoter + GFP: Universal promoter drives a reporter (GFP) in cells where Cre is active.

  2. EGFP in Agtr2 BAC clone: Fluorescent protein inserted into a Bacterial Artificial Chromosome (BAC) to mirror endogenous Agtr2 expression.

  3. Homologous recombination / CRISPR/Cas9: Reporter gene placed next to the endogenous gene for faithful expression.

Strengths and Weaknesses of Reporter Mouse Generation Approaches

Approach

Strengths

Weaknesses

Memorization Aid

CRE/LOX SYSTEM

Good for cell-type specific visualization; flexible.

Potential for false positives (due to leaky Cre expression).

"CLOX-FOX": CreLox can sometimes FOX you (false positives).

GENE TARGETING

Analogous to endogenous levels; less risk of false positives; precise.

Can be more complex to generate.

"GT-Real": Gene Targeting for Real (endogenous) expression.

GENOMIC FRAGMENT

Flexibility and broader utility for various applications.

May not capture all regulatory elements.

"GF-Flex": Genomic Fragment for Flexibility.

TRANSGENE

Simple to generate initially; can achieve high expression.

Limited to specific applications; lacks broad versatility.

"TG-Specific": Transgene is very specific/limited.

Summary of Key Concepts (Reinforcement for Memorization)

  • Transgenic vs. Gene Targeting:

    • Transgenic: Random integration of foreign DNA.

    • Gene Targeting: Precise modification at a specific genomic locus (e.g., homologous recombination).

  • CRISPR/Cas9: A powerful gene editing tool for precise introductions or deletions (e.g., inserting LoxP sites).

  • Conditional Gene Manipulations (Cre/lox): Allows control over when and where a gene is modified.

    • Components: Cre recombinase + LoxP sites.

    • Uses: Deletion, knock-in, reporter expression.

    • Inducible systems: Tamoxifen, Tetracycline for temporal control.

  • Reporter Mouse Strains: Visualize specific cells/gene expression; useful for difficult-to-detect genes and live cell studies.