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

描述（由申请人提供）：本发明描述了一种新的敲入策略，以产生仅表达IIA型前胶原同种型的重组小鼠。该方法的新方面涉及改变II型前胶原基因（Col 2a 1）中受调控外显子的剪接位点，使得外显子将始终包含在最终mRNA中。在这样做时，将利用天然细胞剪接机制，这是重要的，因为越来越多的报道称转录和前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，其中通常将无内含子cDNA引入感兴趣的基因组区域。如果成功的话，我们提出的敲入技术的应用可以用于操纵任何感兴趣的基因内的受调节的外显子的剪接位点序列。因此，将获得关于来自相同基因的不同蛋白质异构体的体内生物学功能的宝贵信息。这在后基因组测序时代特别有吸引力，我们现在知道大多数蛋白质多样性来自前mRNA的选择性剪接。Col 2a 1的选择性剪接是发育调节的，其中外显子2被软骨祖细胞剪接（包括）形成IIA型前胶原，而分化的软骨细胞排除外显子2形成IIB型前胶原。由于这种IIA-IIB开关对软骨发育是特异性的，我们假设Col 2a 1的选择性剪接是软骨正确发育所需的基本承诺机制。我们建议改变外显子2的5'剪接位点处的四个内含子核苷酸，我们已经证明，通过利用COL 2A 1微型基因的体外方法，将导致外显子2的组成性剪接，从而仅产生IIA型前胶原同种型。我们决定在小鼠中表达IIA型，以潜在地避免致死性问题，因为这种同种型也在其他组织如心脏和肾脏的发育早期表达。如果成功的话，我们预测IIA小鼠将含有异常软骨，这是由于通常构成大部分细胞外基质的“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。这种新的模型系统将提供显着的洞察力，在软骨形成过程中的Col 2a 1亚型的生物学功能，也提供了额外的知识与软骨修复机制，因为IIA型前胶原亚型也被证明是在骨关节炎重新表达。

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.