Module_13_Genetic_Engineering_Transgenics_2 2

Germline Knockout Mouse Models

  • Limitations of Classic Germline Knockout Models

    • Embryonic lethality occurs if the targeted gene is crucial for development.

  • Experimental Strategies

    • Cre/loxP System: Developed for

      • Specific deletion in selected tissues.

      • Induction of knockout postnatally (Conditional knockout).

    • Cre Recombinase: Tyrosine recombinase enzyme from P1 bacteriophage performing site-specific recombination between LoxP sites.

Cre/loxP System Details

  • LoxP Sites

    • 34 base pairs (bp) long, consisting of two 13 bp palindromic sequences flanking an 8 bp spacer.

  • Mechanism

    • Cre recombinase recognizes LoxP sites and excises the flanked sequences, leaving a single LoxP site.

    • Crossing a Cre-expressing mouse with a LoxP-flanked gene mouse results in gene deletion in the tissue of interest when the driver is expressed.

Conditional Knockout Generation

  • Mice with LoxP-flanked Gene

    • Created using transgenic embryonic stem cell injection methods; termed "floxed mice" (Flanked Lox).

  • Cre Recombinase Mouse Line

    • Generated using various promoters for tissue-specific expression:

      • Albumin Promoter: Expresses Cre in the liver.

      • Pax7 Promoter: Targets muscle stem cells.

      • Alpha-Myosin Heavy Chain Promoter: Induces Cre in the heart.

Rearrangements by LoxP Sites

  • Types of Genetic Rearrangements

    1. Deletion: If LoxP sites face in the same direction, sequence between them is excised as circular DNA.

    2. Inversion: Opposite orientations of LoxP sites on the same strand lead to inversion of the DNA segment.

    3. Translocation: LoxP sites on separate DNA molecules lead to translocation events.

FLP-FRT System

  • Similar Functionality: Used in mouse-based research, employing flippase (FLP) recombinase from Saccharomyces cerevisiae.

  • FRT Sequences: Recognized by FLP to excise genomic regions of interest.

General vs Conditional Knockout Targeting Vectors

  • Initial Steps in Knockout Generation:

    1. Culturing embryonic stem cells (ES cells) from mouse trophoblasts.

    2. Introducing a replacement vector into ES cells, selecting for homologous recombination through:

      • Positive Selection: Neomycin resistance for recombinant ES cells.

      • Negative Selection: Ganciclovir to eliminate non-homologous recombinants.

Design of Targeting Vectors

  • Basic Structure: Contains 5' and 3' arms homologous to the target gene.

    • Selection Markers: Positive (Neo) and negative (DTA) for gene function disruption.

    • Exon flanked by LoxP sites allows for conditional removal.

Cre Expression Variants

  • Promoter-Regulated Cre: Determines tissue-specific expression (e.g., Ubiquitin C for global expression).

  • Inducible Cre: Requires exogenous ligands (e.g., tamoxifen) for activation, allowing temporal regulation.

    • Examples: MerCreMer and CreERt2 demonstrate this specificity.

Conditional Knockout Summary

  1. Generate mice carrying LoxP-flanked genes through transgenic embryonic stem cell injections.

  2. Create mice expressing the Cre recombinase gene similarly.

  3. Cross the two mouse types to obtain a first-generation Cre-loxP mouse.

Generation of Knockin Mice

  • Purpose: Insert specific DNA sequences at particular genome loci.

  • Examples: MerCreMer and CreERt2, which require tamoxifen for activity.

    • Knocking Strategy: Targeting cassettes introduced upstream of specific genes.