Ming, common genetically engineered mice include gene knockout, conditional gene knockout, gene knock-in (point mutation or fragment insertion), transgenic, targeted transgenic (overexpression), and humanized models.

 

How does their breeding differ from that of ordinary mice?

 

The main differences between breeding genetically engineered mice and ordinary mice lie in breeding strategies, mating schemes, genotype identification, and breeding cycles.

 

Breeding genetically engineered mice requires designing complex mating schemes based on specific gene editing objectives. The breeding cycles are typically longer, often requiring multiple generations of mating and screening to obtain stable genotypes and phenotypes.
 

KO/KI Mouse Breeding

 

Definitions
 

1.  Gene Knockout (KO) Mice: These are mouse models created through genetic engineering techniques that delete or disrupt part or all of a specific gene sequence within the mouse, causing it to lose its original function.

a.  Application: Studying the role of a gene in physiological and pathological processes.

2.  Gene Knock-in (KI) Mice: These are mouse models created through gene editing techniques that precisely insert an exogenous gene sequence into a specific location in the mouse genome, utilizing the mouse's own regulatory elements to guide the expression of the new gene.

a.  Application: Studying gene expression regulation, signaling pathways, and constructing disease models.

KO/KI Mouse Breeding

Population expansion strategies for KO mice include: mating heterozygous (carrier) mice with the gene knockout to wild-type mice to increase genotypic diversity and expand the overall population size; or mating heterozygous mice with each other, aiming to screen for homozygous mice in the offspring for in-depth study of the effects of complete gene knockout.
 

 

Important Notes:
 

● Avoid Cross-line Mating: In the breeding process of gene knockout mice, knockout mice from different lines should not be mated with each other. This is because each line may carry a unique genetic background or modifications. Cross-line mating can introduce unpredictable genetic complexity, affecting experimental results.

● Homozygous Lethality Risk: When heterozygous gene knockout mice are mated with each other, there is a possibility of failing to obtain the expected homozygous mice. This is often because the homozygous state of the gene knockout has a lethal effect on the gene's function, causing homozygous mice to die during development. This is termed homozygous lethality.

The breeding of gene knock-in mice is essentially the same as that for gene knockout mice. However, knock-in mice are not classified into different 'lines'. Therefore, knock-in mice can be mated with each other.

 

Establishing Transgenic Mouse Lines
 

Line Establishment
 

Transgenic mice are animal models created by introducing exogenous genes into mice through genetic engineering techniques, enabling stable inheritance and expression of these exogenous genes.

Breeding Method: Typically involves breeding the same batch of founder (F0) transgenic mice with normal wild-type mice.

● If 20 F1 offspring are obtained and no positive (transgenic) pups are identified, this may indicate the transgene inserted into a silent region, or the transgene is not heritable. In this case, that line can be discarded.

● If F1 offspring are positive, then 2-3 mice from that line are selected and bred with background strain mice to produce the N2 generation.

● If the positive rate in the N2 generation is close to 50%, the line is considered established. Otherwise, continue intercrossing and screening.
 

 

Important Notes:
 

1.  The site of transgene insertion or copy number is different for each founder mouse. Each founder mouse must be bred and studied as a separate line. Founder mice should never be mated with each other.

2.  In the absence of a clear method to distinguish heterozygotes from homozygotes, mating between heterozygotes is strictly prohibited.

 

References:

[1] Peng Xu, Yin Hailin. Breeding strategies for genetically engineered mice. Experimental Science and Technology, 2021, 19(05), 31-37.

[2] Parker-Thornburg, I. (2020). Breeding strategies for genetically modified mice. In: Singh, S.R., Hoffman, R.M. (eds) Mouse Genetics: Methods and Protocols. Methods in Molecular Biology, vol 2066. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9837-1_13

[3] Yu Keke, Wang Siying. Research progress of transgenic mice in life sciences. Medical Review, 2015, 21(1), 23-24.

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