New methods for analysing gene function

Newly developed methods to produce and analyse genetic mosaics are expected to allow researchers to induce multispectral genetic mosaics in vertebrate models such as mice and zebrafish.

The methods have been developed by scientists at the Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) with the findings published in the journal Cell.

The technology is expected to help advance understanding of the function and interaction of genes, at high spatio-temporal resolution, during development and disease. Genes encode the information needed to synthesise proteins, the functional building blocks of our cells. Improved methods for studying gene function will allow researchers to improve knowledge about “how the genome operates in the multiple cell types that make up our bodies and understand gene interaction networks and their regulatory hierarchies”. This knowledge is said to be crucial for designing efficient therapeutic strategies to modify or correct genetic activity in disease.

The ability to modify gene activity has radically changed approaches to the study of biological processes. Lead author Rui Benedito explained that in experimental approaches using inducible genetic mosaics, the induced genetic changes take place only in some cells of the organism (mutant cells), while the other cells remain unaltered (normal cells). “The use of inducible genetic mosaics is very important because it enables us to study how cells with different genotypes behave in the same environment, so that any differences can be attributed to the induced genetic alteration.” This approach to the study of gene function is said to be more precise and informative than the use of classical genetics, in which modification of the target gene in all cells of the organism can generate secondary alterations that cannot be controlled temporally or spatially and that could distort interpretation of the function of the gene in the process under study.

Genetic mosaics have been used extensively in fruit flies due to the ease of performing mitotic recombination and they have revolutionised the study of gene function and cell biology. However, it has so far been technically much more challenging to induce and analyse genetic mosaics in mice, the most widely used model organism in biomedical research. To generate genetic mosaics in mice, scientists at the Molecular Genetics of Angiogenesis lab at the CNIC have developed new molecular biology and transgenesis methods. These methods allow the simultaneous induction, in a single mouse, of multiple mosaic genetic modifications associated with the expression of distinct fluorescent markers that can be detected by high-resolution multispectral microscopy.

To make these new genetic tools usable by other research groups, the CNIC team first developed an open-source DNA engineering strategy that simplifies the production of large and complex DNA constructs containing multiple target genes and fluorescent marker genes in frame. The team also developed new CRISPR/Cas9 methods to introduce these DNA molecules into embryonic stem cells or zygotes, simplifying the rapid and reliable generation of transgenic mouse mosaics.

With these new genetic tools and transgenic mice, the CNIC team was able to study the function of multiple genes in the same tissue by simultaneous fluorescence microscopy of up to 15 colour-coded cell populations, each expressing a unique combination of genes. According to Rui Benedito, “This technology for the first time allows combinations of multiple genetic mosaics to be induced and analysed in different cells of the same mouse. Because the different cell populations are induced in the same tissue, and can be imaged simultaneously, an analysis of how their behaviour differs can provide precise data and new insights into the function of the genes under study. This approach has real advantages over established procedures for genetic analysis, which require comparison of cells present in different tissues or environments, since the analysis must be done with independent animals that have or do not have a given genetic modification.”