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Rosa26 locus gene knockin mouse model

1.Definition

A transgenic mouse model that introduces a gene into the Rosa26 locus. A conventional transgenic mouse model is prepared by pronuclear injection of the plasmid in the zygote, and multiple founders are usually obtained. For different founders, integration loci on the chromosome and plasmids’ copy numbers are different, so it is difficult to get consistent results. The phenotype of the founder would be easy to lose during the transgenic mouse's passage, due to transgene copy number attenuation and gene silencing, which means the experiment cannot be repeated. Currently, a majority of labs prepare transgenic mice by using a fixed-point transgene, and the most commonly used locus is Rosa26.


2. Advantages and disadvantages

Advantages:
Disadvantages:
Definite recombination position of exogenous geneLonger time needed to construct the model compared to a random transgene
Only one copy inserted
Exogenous gene can be expressed in specific tissues at a specified time by combining with the Cre-loxP systemGenetic background is limited by ES cell background
Fewer mice are needed compared with a random transgene method


3.Principle

The Rosa26 gene knockin technology was developed by Phillipe Soriano. There are two exons in the Rosa26 locus, and the exogenous DNA sequence is inserted in between the two exons. In the original design, the upstream of target gene cDNA has a section of transcription termination “STOP” (three copies of SV40 poly A sequence, tPA) sequence. There is one Loxp locus at each end of the transcription termination sequence, and the transcription of the whole structure is controlled by the Rosa26 promoter if insertion occurs in the Rosa26 gene position. Without Cre, the existence of the“STOP” sequence between Rosa26 promoter and target gene prevents target gene expression. If Cre is expressed, the Cre recombinase will remove the “STOP” sequence, and the target gene expression can be promoted by the Rosa26 promoter. Studies have shown that the Rosa26 gene can code a non-essential nuclear RNA in almost all tissues, and is an inserting hotspot of the exogenous gene. The Rosa26 locus gene inserting technology can construct all-purpose conditional transgenic mouse models effectively, and has become a favorite of several researchers. However, the promoting ability of the Rosa26 promoter is limited in some tissues, so the target gene usually cannot meet the expression level required by researchers. To solve this problem, the original Rosa26 gene knockin system was improved, and a strong promote, such as the CMV promoter fused by the chicken actin promoter and CMV enhancer. is introduced, and the expression of target gene can be promoted highly effectively by replacing the Rosa26 promoter with the hybrid promoter. The gene knockin strategy is shown in the following figure:

 

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4.Application

Gene overexpression study

Expression and functional study of the exogenous gene in the mouse


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