Comparative transcriptomic analysis of the proximal tibial growth plates of 14-day old Longshanks and wildtype mice

This study was undertaken to uncover the molecular pathways that underlie differences among individuals in rates of longitudinal growth and mature length of the long bones in a rodent model. To do so, the transcriptomic profiles of the proximal epiphyses of 14-day old CD-1 wildtype mice and the Longshanks mice were compared. The Longshanks mouse was selectively bred for longer tibiae in relation to body mass (see Marchini et al 2014 – BMC Evol Biol 14:258). Over 13 generations, Longshanks tibiae were 11-12% longer compared to random-bred wildtype mice (hereafter Controls) from the same genetic background (ICR or CD-1), but body mass was unchanged. By comparing the transcriptomes of samples of Control and Longshanks proximal tibiae, we sought to identify differentially expressed genes that were associated with the faster growth and longer tibia of the Longshanks mouse, as a window into the pathways that may underlie intra- and interspecific differences in limb bone size and shape. Results were further validated using qPCR and cell and tissue culture assays. Three Longshanks mice (line LS1, biological replicates) and three Control mice (biological replicates) from different families in generation F13 were used for RNA sequencing at 14 days old. The two tibial proximal epiphyses from each mouse were pooled for total RNA extraction, sequencing and differential gene expression analyses as described in the protocols.