Droplet digital PCR or quantitative PCR for in-depth genomic and functional validation of genetically altered rodents

Loic Lindner , Pauline Cayrou , Thomas W Rosahl , Heather H Zhou , Marie-Christine Birling , Yann Herault , Guillaume Pavlovic (1, 2)

1 PHENOMIN - French National Infrastructure for Mouse Phenogenomics
2 IGBMC - Institut de Génétique et de Biologie Moléculaire et Cellulaire

Gene targeting and additive (random) transgenesis have proven to be powerful technologies with which to decipher the mammalian genome. With the advent of CRISPR/Cas9 genome editing, the ability to inactivate or modify the function of a gene has become even more accessible. However, the impact of each generated modification may be different from what was initially desired. Minimal validation of mutant alleles from genetically altered (GA) rodents remains essential to guarantee the interpretation of experimental results. The protocol described here combines design strategies for genomic and functional validation of genetically modified alleles with droplet digital PCR (ddPCR) or quantitative PCR (qPCR) for target DNA or mRNA quantification. Indepth analysis of the results obtained with GA models through the analysis of target DNA and mRNA quantification is also provided, to evaluate which pitfalls can be detected using these two methods, and we propose recommendations for the characterization of different type of mutant allele (knock-out, knock-in, conditional knockout , FLEx, IKMC model or transgenic). Our results also highlight the possibility that mRNA expression of any mutated allele can be different from what might be expected in theory or according to common assumptions. For example, mRNA analyses on knockout lines showed that nonsense-mediated mRNA decay is generally not achieved with a critical-exon approach. Likewise, comparison of multiple conditional lines crossed with the same CreER T2 deleter showed that the inactivation outcome was very different for each conditional model. DNA quantification by ddPCR of G0 to G2 generations of transgenic rodents generated by pronuclear injection showed an unexpected variability, demonstrating that G1 generation rodents cannot be considered as established lines.

Domaines: Biochimie, Biologie Moléculaire

Loic Lindner, Pauline Cayrou, Thomas W Rosahl, Heather H Zhou, Marie-Christine Birling, et al.. Droplet digital PCR or quantitative PCR for in-depth genomic and functional validation of genetically altered rodents. Methods, 2021, 191, pp.107 - 119. ⟨10.1016/j.ymeth.2021.04.001⟩. ⟨hal-03306827⟩