Gurley Kyle A, Chen Hao, Guenther Catherine, Nguyen Elizabeth T, Rountree Ryan B, Schoor Michael, Kingsley David M
Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
J Bone Miner Res. 2006 Aug;21(8):1238-47. doi: 10.1359/jbmr.060515.
To reveal the ANK complete loss of function phenotype in mice, we generated conditional and null alleles. Mice homozygous for the null allele exhibited widespread joint mineralization, similar in severity to animals harboring the original ank allele. A delayed yet similar phenotype was observed in mice with joint-specific loss of ANK function.
The ANK pyrophosphate regulator was originally identified and proposed to play a key role in articular cartilage maintenance based on a single spontaneous mouse mutation (ank) that causes severe generalized arthritis. A number of human mutations have subsequently been reported in the human ortholog (ANKH), some of which produce skull and long bone defects with no apparent defects in joints or articular cartilage. None of the currently known mouse or human mutations clearly eliminate the function of the endogenous gene.
Two new Ank alleles were generated using homologous recombination in mouse embryonic stem (ES) cells. Joint range of motion assays and muCT studies were used to quantitatively assess phenotypic severity in wildtype, heterozygous, and homozygous mice carrying either the null (Anknull) or original (Ankank) allele. A Gdf5-Cre expressing line was crossed to mice harboring the conditional (Ankfloxp) allele to eliminate ANK function specifically in the joints. Histological stains and beta-galactosidase (LACZ) activity were used to determine the correlation between local loss of ANK function and defective joint phenotypes.
Anknull/Anknull mice develop severe ectopic postnatal crystal deposition in almost every joint of the body, leading to eventual joint fusion and loss of mobility. The severity of phenotype in these mice is indistinguishable from that of Ankank/Ankank mice. In addition, despite the widespread expression of Ank in many tissues, the specific deletion of Ank in joints also produces joint mineralization and ankylosis.
These studies show that ANK function is required locally in joints to inhibit mineral formation and that the Ank gene plays a key role in postnatal maintenance of joint mobility and function.
为了揭示ANK在小鼠中的完全功能丧失表型，我们构建了条件性和无效等位基因。无效等位基因的纯合小鼠表现出广泛的关节矿化，其严重程度与携带原始ank等位基因的动物相似。在ANK功能关节特异性缺失的小鼠中观察到延迟但相似的表型。
ANK焦磷酸盐调节因子最初是基于一种导致严重全身性关节炎的自发小鼠突变（ank）而被鉴定出来，并被认为在关节软骨维持中起关键作用。随后在人类同源基因（ANKH）中报道了许多人类突变，其中一些会导致颅骨和长骨缺陷，但关节或关节软骨没有明显缺陷。目前已知的小鼠或人类突变均未明确消除内源性基因的功能。
利用小鼠胚胎干细胞（ES）中的同源重组构建了两个新的Ank等位基因。使用关节活动范围测定和微计算机断层扫描（muCT）研究来定量评估携带无效（Anknull）或原始（Ankank）等位基因的野生型、杂合子和纯合子小鼠的表型严重程度。将表达Gdf5-Cre的品系与携带条件性（Ankfloxp）等位基因的小鼠杂交，以特异性消除关节中的ANK功能。使用组织学染色和β-半乳糖苷酶（LACZ）活性来确定ANK功能局部丧失与关节缺陷表型之间的相关性。
Anknull/Anknull小鼠在出生后几乎身体的每个关节都出现严重的异位晶体沉积，最终导致关节融合和活动丧失。这些小鼠的表型严重程度与Ankank/Ankank小鼠无法区分。此外，尽管Ank在许多组织中广泛表达，但关节中Ank的特异性缺失也会导致关节矿化和强直。
这些研究表明，关节局部需要ANK功能来抑制矿物质形成，并且Ank基因在出生后关节活动度和功能的维持中起关键作用。
