Purpose

The retina of the eye plays the role of a sensor that converts the light from the outside into an electrochemical signal and sends it to the brain. The retina is composed of photoreceptor cells that accept light, retinal pigment epithelial cells that support it, and various nerve cells that transmit signals to the brain. Abnormalities in these cells cause visual impairment. The purpose of this research was to provide all essential genes necessary for biochemical, molecular biological, and genetic studies of the retina. Specific goals are as follows. Specific goals are the following two points.

(1) All genes expressed in retinal cells are collected in the form of full-length cDNA.

(2) As causative gene candidates causing retinal degeneration, groups of genes specifically expressed in retinal cells are identified.

The full-length cDNA collection of the obtained human retina-derived gene is useful for elucidating the mechanism of degeneration and regeneration of retinal cells and for elucidating the cause of hereditary retinal degenerative diseases.

Development of full length cDNA library preparation method

In order to obtain all genes expressed in retinal cells, we have developed a novel full-length cDNA synthesis method. This method, named vector capping method (V-capping method), has the following features.

  1. It consists of only three steps.
  2. Since PCR process and restriction enzyme treatment process are not included, artificial mutation or deletion does not occur.
  3. A library consisting of 106 independent clones can be generated from a few μg of total RNA.
  4. Due to the presence of extra G added to the 5' terminal, it can be determined whether it is a full-length cDNA.
  5. A library with a content of full-length cDNA clones of 95% or more can be prepared.
  6. It is possible to prepare a size-bias free library containing full-length cDNA clones up to 13 kbp.

Cloning of retinal cell-specific genes

Using the V-capping method, a full-length cDNA library was prepared from the photoreceptor-derived cell line Y79 and the retinal pigment epithelial cell line ARPE-19. Tens of thousands of clones are picked up from each library. We have identified genes that are expressed specifically or preferentially in each cell. Among them, many new variants were included, which are different from the known transcription start sites and splicing sites.

Later, about 160,000 full-length cDNA clones isolated so far have been released at the Riken BioResource Center DNA Bank.

References

  1. Kato S, Ohtoko K, Ohtake H, Kimura T., Vector-capping: a simple method for preparing a high-quality full-length cDNA library., DNA Res. (2005) 12:53-62.
  2. Oshikawa M, Sugai Y, Usami R, Ohtoko K, Toyama S, Kato S. Oshikawa M, Sugai Y, Usami R, Ohtoko K, Toyama S, Kato S., Fine expression profiling of full-length transcripts using a size-unbiased cDNA library prepared with the vector-capping method., DNA Res. (2008) 15:123-36.
  3. Oshikawa M, Usami R, Kato S., Characterization of the arylsulfatase I (ARSI) gene preferentially expressed in the human retinal pigment epithelium cell line ARPE-19., Mol Vis. (2009) 15:482-94.
  4. Kato S, Oshikawa M, Ohtoko K., Full-length transcriptome analysis using a bias-free cDNA library prepared with the vector-capping method., Methods Mol Biol. 2011;729:53-70.
  5. Oshikawa M, Tsutsui C, Ikegami T, Fuchida Y, Matsubara M, Toyama S, Usami R, Ohtoko K, Kato S., Full-length transcriptome analysis of human retina-derived cell lines ARPE-19 and Y79 using the vector-capping method., Invest Ophthalmol Vis Sci. (2011) 52:6662-6670.