Alizadeh Azita, Clark John, Seeberger Teri, Hess John, Blankenship Tom, FitzGerald Paul G
Department of Cell Biology and Human Anatomy, University of California, Davis, CA 95616, USA.
Invest Ophthalmol Vis Sci. 2004 Mar;45(3):884-91. doi: 10.1167/iovs.03-0677.
The 129 strain of mouse carries a mutation in the gene for CP49 (phakinin), an intermediate filament protein thus far demonstrated only in the lens fiber cell. As such, these mice represent naturally occurring mutants of interest in the study of the lens cytoskeleton. However, this strain of mouse is also widely used as a source of embryonic stem cells in gene-targeting studies. The presence of a mutation in a lens-specific gene can confound interpretation of studies in which lens genes have been knocked out. In the present study, both the genotype and phenotype of these mice were characterized, to permit an evaluation of the biological impact of this mutation and to facilitate the discrimination between wild-type and mutant animals that have been derived from this strain in gene-targeting studies.
The CP49 cDNA and, when relevant, the genomic DNA sequences were determined for the 129/SvJ and C57BL/6J mice and from a commercially available 129/OLa P1 genomic clone. PCR primers were screened for their capacity to discriminate between the mutant and wild-type CP49 genes. Northern blot analysis was used to assess mRNA levels for CP49, filensin, and gammaS-crystallin (control). Western blot analysis was used to identify changes in protein size and abundance. The impact of the mutation on lens architecture was evaluated at the light-microscope level. Lens fiber cell ghosts from mutant and wild-type mice were examined in the electron microscope for the presence of beaded filaments. Lens clarity was assessed by slit lamp.
The 129 strain of mice exhibited a 6303-bp deletion from the end of intron B, and the beginning of exon 2. This deletion results in the loss of the exon 2 splice acceptor site, absence of exon 2 from the CP49 mRNA, and dramatically reduced levels of CP49 mRNA. The CP49 protein was undetectable by Western blot analysis. Messenger RNA levels for filensin, CP49's assembly partner, were normal, but protein levels were sharply reduced. Light microscopy established that the initial differentiation and elongation of the fiber cells proceeded normally. Electron microscopy showed the absence of beaded filaments, whereas slit lamp microscopy showed a slowly emerging and progressive loss of optical clarity.
The 129/SvJ and 129/OLa strains of mice harbor a mutation that sharply reduces CP49 mRNA levels and essentially eliminates both CP49 and the beaded filament. These lenses exhibited a slow but progressive loss of optical clarity with age. Thus, the 129 strain of mouse behaves as a functional CP49 knockout. The loss of clarity in the lenses of these animals and the absence of beaded filaments (and any attendant interactions that may exist between beaded filaments and other lens proteins/structures) suggest that gene-targeting studies of lens proteins in which the 129 strain was used as a source of embryonic stem cells may need reevaluation.
129品系小鼠的CP49（phakinin）基因发生了突变，CP49是一种中间丝蛋白，迄今为止仅在晶状体纤维细胞中被证实存在。因此，这些小鼠是研究晶状体细胞骨架的天然突变体。然而，该品系小鼠也被广泛用作基因靶向研究中胚胎干细胞的来源。晶状体特异性基因中的突变可能会混淆对敲除晶状体基因的研究结果的解释。在本研究中，对这些小鼠的基因型和表型进行了表征，以评估该突变的生物学影响，并便于在基因靶向研究中区分源自该品系的野生型和突变型动物。
测定了129/SvJ和C57BL/6J小鼠以及从市售的129/OLa P1基因组克隆中获得的CP49 cDNA及相关基因组DNA序列。筛选了能够区分突变型和野生型CP49基因的PCR引物。采用Northern印迹分析评估CP49、丝状晶状体蛋白和γS-晶状体蛋白（对照）的mRNA水平。采用Western印迹分析鉴定蛋白质大小和丰度的变化。在光学显微镜水平评估突变对晶状体结构的影响。在电子显微镜下检查突变型和野生型小鼠的晶状体纤维细胞幽灵，以确定是否存在串珠状细丝。通过裂隙灯评估晶状体透明度。
129品系小鼠在内含子B末端和外显子2起始处有一个6303 bp的缺失。该缺失导致外显子2剪接受体位点的丢失，CP49 mRNA中缺少外显子2，且CP49 mRNA水平显著降低。Western印迹分析未检测到CP49蛋白。CP49的组装伴侣丝状晶状体蛋白的mRNA水平正常，但蛋白水平急剧降低。光学显微镜检查表明纤维细胞的初始分化和伸长过程正常。电子显微镜检查显示不存在串珠状细丝，而裂隙灯显微镜检查显示光学清晰度逐渐缓慢丧失。
129/SvJ和129/OLa品系小鼠存在一种突变，该突变显著降低了CP49 mRNA水平，并基本消除了CP49和串珠状细丝。随着年龄的增长，这些晶状体的光学清晰度逐渐缓慢丧失。因此，129品系小鼠表现为功能性CP49基因敲除。这些动物晶状体透明度的丧失以及串珠状细丝的缺失（以及串珠状细丝与其他晶状体蛋白/结构之间可能存在的任何相关相互作用）表明，以129品系作为胚胎干细胞来源的晶状体蛋白基因靶向研究可能需要重新评估。
