Question:  What are conditional knockout mice, and how are they different from gene knockout mice?

Answer:  Let's first explain the differences between the two.

 

01. Differences Between KO and CKO
 

Conditional Gene Knockout (CKO):  Refers to knocking out the target gene in specific tissues or at specific time points in mice.

●Advantages:  Overcomes embryonic lethality or premature death resulting from the complete knockout of essential genes. CKO mice can accurately simulate the onset and progression of human diseases, making them ideal models for studying gene function.

●Disadvantages:  The mating process is relatively complex; the construction cycle for the mouse model is relatively long.

Whole-Body Gene Knockout (KO):  Refers to knocking out the target gene in all tissues and cells of the mouse, suitable for studying the global function of a gene.

●Advantages:  Short construction cycle for the mouse model; simple mating process.

●Disadvantages:  Approximately one-third of genes, when knocked out homozygously, lead to embryonic or perinatal death in mice, limiting their application in certain research areas.

 

Question:  How can we obtain conditional knockout mice?
 

02. Constructing Conditional Knockout Mice Using the Cre-loxP System

The core of the Cre-loxP system lies in the specific recognition between Cre recombinase and loxP sites:

●loxP Sites:  Inserted on both sides of the target gene, marking the gene region to be knocked out.

●Cre Recombinase:  Expressed in specific tissues or at specific times. It recognizes and cuts the DNA between two loxP sites, achieving gene knockout.

●Floxed Mouse (flox):  A mouse model with loxP sites inserted on both sides of the target gene. The target gene retains normal function in the absence of Cre recombinase.

●Cre Driver Mouse:  A mouse model that expresses Cre recombinase in specific tissues or at specific times.

By breeding a floxed mouse with a Cre driver mouse, Cre recombinase will recognize and cut the DNA between the loxP sites in the specific tissue or at the designated time, thereby achieving tissue-specific or temporal knockout of the target gene.

 

03. Construction Process

1.Construct the floxed mouse:  Insert loxP sites on both sides of the target gene to construct the floxed mouse.

2.Select the Cre driver mouse:  Based on experimental needs, choose a Cre driver mouse that expresses Cre recombinase in the specific tissue or cell type.

3.Breeding and Screening:

a.Breed the F0 generation carrying the loxP sites with wild-type mice to obtain F1 heterozygotes.

b.Interbreed F1 heterozygotes (flox/+) to obtain F2 homozygotes (flox/flox).

c.Breed F2 homozygotes (flox/flox) with whole-body/tissue-specific Cre driver mice to obtain the F3 generation (flox/+, Cre).

d.Interbreed the F3 generation (flox/+, Cre) to obtain the F4 generation (flox/flox, Cre), which are the homozygous CKO mice.

Important Considerations:

1.Genetic linkage issue:  Ensure the Cre and flox genes are located on different chromosomes to avoid linkage.

2.Inducer usage:  If using Tamoxifen to induce the CreERT2 system, pay attention to dosage and administration method to avoid cross-contamination.

3.Validation of knockout efficiency:  Validate successful gene knockout using methods such as PCR, Western blot, etc.

 

Question:  What are the advantages of constructing conditional knockout mice using the Cre-loxP system?
 

04. Advantages of the Cre-loxP System

1.Spatiotemporal Specificity:  Enables gene knockout in specific tissues or at specific times.

2.High Efficiency:  Cre recombinase has high activity, with recombination efficiency reaching over 70%.

3.Flexibility:  Can be combined with various promoters to achieve a wide range of spatiotemporal control.

Conditional knockout mice are widely used in disease model construction, gene function research, and drug development.

●Cancer Research:  Activating or knocking out oncogenes in specific tissues (e.g., breast, lung, colon) to simulate tumor initiation and development. For example, studying the impact on breast cancer development by inducing knockout of a tumor suppressor gene specifically in mouse mammary cells.

●Cardiovascular Disease:  Knocking out specific genes in heart tissue to study their contribution to myocardial infarction, heart failure, and other cardiovascular diseases.

●Neuroscience:  Simulating pathological changes of neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease). For example, studying the effects on memory and cognitive function by specifically knocking out certain genes in the mouse hippocampus.

 

References:

[1] Relevant article on conditional knockout mice.

[2] Kim, H., Kim, M., Im, S. K., & Fang, S. (2018). Mouse Cre-LoxP system: general principles to determine tissue-specific roles of target genes. Lab. Anim. Res., 34(4), 147-159.

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