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X. Q. Guo, J. D. Zieske; Cloning of Type I and V Collagen Genes Into a Retroviral Vector. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6208.
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© ARVO (1962-2015); The Authors (2016-present)
Our laboratory has developed a 3-dimensional (3D) culture system of human corneal fibroblasts (HCFs) to allow us to examine the synthesis and deposition of extra cellular matrix (ECM). In order to study the assembly of collagens type I and V into fibrils deposited into the ECM, we are trying to clone these genes into HCFs.
Both type I and type V collagens have 2 chains: α1 and α2, and their gene sizes are 4395bp and 4101bp (type I α1 and α2, respectively), and 5517bp and 4500bp (type V α1 and α2, respectively). The genes of interest were cut into pieces and RT-PCR was performed using specific primers. The PCR product was digested with specific restriction enzymes, separated on a gel, purified, ligated together to form the entire gene, and inserted into retroviral vectors: pRetroX-IRES-DsRed (α1 chains of Collagen I and V) and pRetroX-IRES-GFP (α2 chains of Collagen I and V).
Several difficulties were encountered in the attempt to clone the whole gene into a vector by a single RT-PCR. These difficulties included the large size of the genes and the similarities of the sequences between different collagens. To resolve these difficulties, the genes were cloned in up to 4 pieces. In addition, primers of up to 36bp were used. Ultimately, all genes were successfully cloned.
Packaging the collagen type I and type V genes into retroviral vectors will allow us to introduce these genes into human corneal fibroblasts. These vectors will allow the examination of ECM assembly in a 3D culture model.
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