GENERATION OF A NOVEL RECOMBINANT MOUSE MODEL EXPRESSING ONLY ONE COL2A1 ISOFORM

描述(由申请人提供)：本提案描述了一种新的敲入策略，以产生仅表达IIA型前胶原异构体的重组小鼠。这种方法的新方面包括改变II型前胶原基因(Col2a1)中受调节的外显子的剪接位置，使该外显子始终包括在最终的mRNA中。在这样做的过程中，将利用自然的细胞剪接机制，这一点很重要，因为越来越多的报道称，转录机制和前mRNA剪接机制在细胞核中紧密协调。我们的模型代表了其他重组“敲入”策略的重要替代方案，在这些策略中，无内含子的cDNA通常被引入到感兴趣的基因组区域。如果成功，我们建议的敲入技术的应用可以用来操纵任何感兴趣基因内受调控的外显子的剪接位点序列。因此，从同一基因衍生的不同蛋白质亚型的体内生物学功能的宝贵信息将被获得。在这个后基因组测序时代，这一点尤其吸引人，因为我们现在知道，大多数蛋白质多样性是由前mRNA的选择性剪接产生的。COL2a1的选择性剪接受发育调节，其中外显子2由形成IIA型前胶原的软骨前体细胞剪接(包括)，而分化的软骨细胞不包括形成IIB型前胶原的外显子2。由于这种IIA-IIB开关是软骨发育所特有的，我们推测COL2a1的选择性剪接是软骨正确发育所必需的基本承诺机制。我们建议改变外显子2 5‘剪接点的4个内含子核苷酸，通过体外方法利用COL2A1微基因，将导致外显子2的结构性剪接，从而只产生IIA型前胶原异构体。我们决定在小鼠中表达IIA型基因，以潜在地避免致命性问题，因为这种异构体也在心脏和肾脏等其他组织的发育早期表达。如果成功，我们预测IIA小鼠将包含异常的软骨，这是由于正常情况下构成细胞外基质的主要成分的“成熟”软骨IIB型胶原纤维缺乏所致。随后，长骨的形成也可能受到影响。这个新的模型系统将为深入了解Col2a1亚型在软骨形成过程中的生物学功能提供重要的信息，并提供与软骨修复机制相关的额外知识，因为IIA型前胶原亚型也被证明在骨关节炎期间重新表达。

DESCRIPTION (provided by applicant): The present proposal describes a novel knock-in strategy to generate a recombinant mouse expressing the type IIA procollagen isoform only. The novel aspect of this approach involves altering the splice site of the regulated exon in the type II procollagen gene (Col2a1) such that the exon will always be included in the final mRNA. In doing so, the natural cellular splicing mechanisms will be utilized which is important given the increasing reports that mechanisms of transcription and pre-mRNA splicing are tightly co-ordinated in the nucleus. Our model represents an important alternative to other recombinant "knock-in" strategies where intron-less cDNAs are commonly introduced into the genomic area of interest. If successful, application of our proposed knock-in technique can be used to manipulate splice site sequences of a regulated exon within any gene of interest. Therefore, invaluable information on the in vivo biological function of different protein isoforms derived from the same gene will be gained. This is particularly appealing in this post-genome sequencing era where we now know that the majority of protein diversity results from alternative splicing of pre-mRNA. Alternative splicing of Col2a1 is developmentally-regulated where exon 2 is spliced (included) by chondroprogenitor cells forming type IIA procollagen while differentiated chondrocytes exclude exon 2 forming type IIB procollagen. As this IIA-IIB switch is specific to cartilage development, we hypothesize that alternative splicing of Col2a1 is an essential commitment mechanism required for correct development of cartilage. We propose to alter four intronic nucleotides at the 5' splice site of exon 2 that we have shown, by in vitro methods utilizing a COL2A1 mini-gene, will result in constitutive splicing of exon 2, thereby only producing the type IIA procollagen isoform. We decided to express type IIA in mice to potentially avoid lethality problems since this isoform is also expressed early during development of other tissues such as heart and kidney. If successful, we predict that the IIA mouse will contain abnormal cartilage due to a deficiency in the "mature" cartilage type IIB collagen fibers that normally make up the majority of the extracellular matrix. Subsequently, long bone formation may also be affected. This novel model system will provide significant insight into the biological function of the Col2a1 isoforms during chondrogenesis and also provide additional knowledge related to cartilage repair mechanisms since the type IIA procollagen isoform has also been shown to be re-expressed during osteoarthritis.