Li S W, Takanosu M, Arita M, Bao Y, Ren Z X, Maier A, Prockop D J, Mayne R
Center for Gene Therapy, MCP Hahnemann University, Philadelphia, Pennsylvania, USA.
Dev Dyn. 2001 Oct;222(2):141-52. doi: 10.1002/dvdy.1178.
Transgenic mice were prepared by homologous recombination with a Col11a2 targeting gene in which an inverted neomycin-resistant gene was inserted between restriction sites in exons 27 and 28. The targeted allele was transcribed in shortened mRNAs, which could be detected by Northern blotting. However, translation of the full-length Col11a2 chain was unable to occur because of the presence of premature termination codons within the inverted neomycin-resistant gene. Analysis of pepsin-resistant collagen chains from rib cartilage of homozygous mice demonstrated the lack of synthesis of intact alpha2(XI) chains. However, pepsin-resistant collagen chains of either alpha1(XI) or alpha1(V) were still detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Therefore, alpha2(XI) chains are not essential for the assembly of some molecular forms of triple-helical type V/XI collagen. The phenotype was milder than in the cho/cho mouse in which, as the result of mutation, translation of the full-length alpha1(XI) chain fails to occur and the mice die at birth (Li et al., 1995). Homozygous mice without expression of an alpha2(XI) chain had a smaller body size, receding snouts, and deafness. Nasal bones in the homozygous transgenic mice were specifically shorter and dimpled on their external surfaces. Chondrocytes in growth plates of all long bones were markedly disorganized and failed to align in columns. Analysis of growth plates from transgenic mice by in situ hybridization showed expression of alpha1(II) and alpha1(XI) but not of alpha1(I) or alpha1(V) which, in contrast, were expressed in the developing bone and in the bone collar. Expression of alpha1(X) specifically in the hypertrophic cartilage was observed in normal and transgenic mice. No obvious osteoarthritis was observed throughout the life of homozygous mice up to 1 year of age, although minor morphologic anomalies in the articular cartilages were discernible. The mild phenotype is consistent with similar mutations in the COL11A2 gene seen in patients with nonocular Stickler syndrome and some patients with otospondylomegaepiphyseal dysplasia (OSMED), as well as in patients with a nonsyndromic form of deafness called DFNA13.
通过用Col11a2靶向基因进行同源重组制备转基因小鼠，该靶向基因中一个反向新霉素抗性基因插入到外显子27和28的限制性位点之间。靶向等位基因转录成缩短的mRNA，可通过Northern印迹法检测到。然而，由于反向新霉素抗性基因内存在提前终止密码子，全长Col11a2链无法进行翻译。对纯合小鼠肋软骨中胃蛋白酶抗性胶原链的分析表明缺乏完整的α2(XI)链的合成。然而，在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上仍可检测到α1(XI)或α1(V)的胃蛋白酶抗性胶原链。因此，α2(XI)链对于某些三螺旋V/XI型胶原分子形式的组装不是必需的。该表型比cho/cho小鼠的表型轻，在cho/cho小鼠中，由于突变，全长α1(XI)链无法翻译，小鼠出生时死亡（Li等人，1995年）。不表达α2(XI)链的纯合小鼠体型较小，口鼻部后缩，且耳聋。纯合转基因小鼠的鼻骨特别短，其外表面有凹痕。所有长骨生长板中的软骨细胞明显紊乱，无法排列成柱状。通过原位杂交对转基因小鼠生长板的分析显示α1(II)和α1(XI)表达，但α1(I)或α1(V)不表达，相反，α1(I)和α1(V)在发育中的骨骼和骨环中表达。在正常和转基因小鼠中均观察到α1(X)特异性在肥大软骨中表达。在1岁以下的纯合小鼠的整个生命过程中未观察到明显的骨关节炎，尽管在关节软骨中可辨别出轻微的形态异常。这种轻度表型与非眼部Stickler综合征患者、一些耳脊椎骨肥大发育异常（OSMED）患者以及一种称为DFNA13的非综合征性耳聋患者中COL11A2基因的类似突变一致。
