High-Throughput Approaches for Generating Targeted Mutations in Mice on a Genome-Wide Scale
The Human Genome Project (HGP) revealed that there were at least 30,000 genes in the mammalian genome. Most of these genes were expressed in the central nervous system (CNS) and usually produce multiple transcripts, encoding different protein products. By extrapolating studies on invertebrates or comparing sequences based on homology, more biochemical properties of these products can be guessed, but it was difficult to predict in vivo functions. Therefore, the use of genomic methods to advance post-genomic research on CNS development and function will help to understand the molecular mechanisms of the nervous system operating in intact organisms.
Why Choose Mice as a Model for Targeted Mutations?
In the past two decades, mice have played an increasingly prominent role in biomedicine, and the proportion of neuroscience research using mice has risen from about 20% to about 50%. This change was mainly due to:
- Mice provide a larger genetic toolbox, especially gene targeting technology based on embryonic stem cells (ES).
- The smaller the size of the mouse brain, the easier is it for light to pass through and reach deeper brain regions, suitable for neurogenetics research.
- Mice are light in weight and have obvious advantages in drug development.
High-Throughput Approaches for Generating Targeted Mutations
Mutation or deletion of endogenous genes in engineered mice is one of the most effective methods to determine gene function, but this method is limited to the generation of "gene targeting vectors" and the selection of rare ES cell clones. The initial use of chemical mutagenesis to generate random mutations or gene traps overcomes the difficulty of breeding mice with specific genetic changes and subsequently developed a high-throughput method capable of manipulating one or more genes of interest - VelociGene.
Fig.1 BAC-based mouse gene targeted mutation method. (Valenzuela, 2003)
VelociGene is a high-throughput and mostly automated process that uses targeting vectors based on bacterial artificial chromosomes (BACs). It allows gene changes with nucleotide precision, is not limited by the size of the deletion required, does not rely on homology or positive or negative selection, and can accurately replace the interest of interest with fragments that allow high-resolution localization of target gene expression Gene.
Just as the development of automated DNA sequencers has made large-scale work of sequencing the entire genome successfully carried out, high-throughput methods are critical to endow the gene functions carried by certain interesting gene fragments in the model organism genome, especially in the field of neuroscience research. The development of the VelociGene method and the effective production of BAC transgenic mice has enabled the analysis of brain gene expression and function to be achieved in vivo, which cannot be achieved by traditional methods. These strategies reveal the function of a single gene in the nervous system and will accelerate the use of functional genomics methods for neuroscience research.
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- Valenzuela, D.M.; et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nature biotechnology. 2003, 21(6): 652-659.