In 1996 Tsumaki and colleagues [
11,
12] studied collagen α2(XI) (
Col11a2) expression and characterized which regulatory elements were required for chondrocyte/cartilage-specific expression
in vivo. They showed that the upstream 742 bp and intron 1 were crucial for expression in primordial cartilage. Shortening the promoter region resulted in less active and more variable expression patterns amongst founder embryos. Deletion of the upstream 290 bp of the 742 bp construct resulted in divergent expression as the primordial cartilage of carpals, tarsals and vertebral bodies no longer expressed LacZ that was cloned downstream of the promoter, whereas notochord and other primordial cartilage were unaffected [
11]. Intron 1 was crucial as it contained an enhancer element important for strong chondrocyte-specific expression, which was also investigated in mouse and chick embryo chondrocytes derived from sterna of 15-day-old embryos [
2,
13]. This was confirmed by Liu and colleagues [
14], who used electrophoretic mobility shift assays (EMSAs) with RCS versus Balb/3 T3 and undifferentiated ATDC5 cells to show that this was mediated through SOX9 transcriptional activation. Tsumaki and colleagues [
11] identified four different cell-specific elements in the
Col11a2 promoter: an element between -742 and -453 bp promoting high level gene expression in most chondrocytes; an element within the 2.3 kb intron 1 necessary for high level expression in notochord and nucleus pulposus; an element in intron 1 directing high expression in most chondrocytes in cooperation with the -453 bp promoter; and finally an element in which intron 1 cooperated with an element between positions -742 and -453 that specifically directed expression in primordial cartilage of carpals, tarsals and vertebral bodies [
11]. Later, others identified that a 1,064 bp promoter fragment resulted in expression not only in chondrocytes, but also in osteoprogenitors in mouse embryos [
15]. Tsumaki and colleagues [
16] showed that, depending on the deleted region of the
Col11a2 promoter, it was possible to activate expression in neural tissues or even other random tissues, thereby further discriminating between a cartilage-specific element (-501 to -530) and a neural tissue-specific element. In 2005 Fujimuki and colleagues [
17] reported that their
Col11a2-cre mouse did not show expression in non-cartilage tissues, including heart, brain and osteoblasts except for the notochord. They stated that the expression level of
Col2a1 is higher, but that the
Col11a2-cre mouse showed higher specificity, potentially due to the lower expression levels.