The endogenous TBPH coding region is replaced with fly codon-optimized Hsap\TARDBP cDNA with an N-terminal Tag:FLAG tag and a Disc\RFPDsRed.3xP3.cUa marker flanked by loxP sites at the C-terminal. The Hsap\TARDBP cDNA contains an ALS-associated sporadic mutation (G294A) and is expressed under the control of the endogenous TBPH regulatory regions.
TBPHKO.hTARDBP.G294A.DsR+ homozygotes of either sex are viable and display no significant changes in developmental timing or longevity compared to controls, but the females show significantly decreased fecundity. Although larval locomotion (distance travelled in 5min) is not perturbed and no defects are detected in the adult daily activity pattern, the total locomotor activity per day is significantly increased relative to controls and the mutant flies also display more rapid age-related decline in climbing ability. The fraction of TBPH aggregates/puncta localized in the cytoplasm (rather than the nucleus) is increased in neurons in the TBPHKO.hTARDBP.G294A.DsR+ adult brain compared to flies carrying the wild-type TBPHKO.hTARDBP.WT.DsR+ allele, however these puncta do not appear to be stress granules due to the lack of the Fmr1 protein marker signal.
