Identification of Differentially Expressed Transcripts of Tdrd7 Null Mutant Mouse Lens
University of Delaware
The ocular lens is a transparent tissue within the eye that focuses light on the retina and facilitates high-resolution vision. Cataract is an eye disease that results due to the loss of lens transparency and causes severely impaired vision. It can be caused by genetic perturbations,aging or physiological conditions like diabetes. Cataract treatment depends upon the patient's specific visual needs and often involves surgery. Over 77 million individuals are affected worldwide, and in the United States alone, costs exceed $3 billion annually. Recently, a mutation in the human TDRD7 (Tudor domain containing protein 7) gene was shown to cause congenital/pediatric cataracts in patients. Moreover, it was also demonstrated that Tdrd7 targeted germline knockout or ENU-induced knockout mouse mutants exhibit cataracts that closely resemble the human phenotype. Previous data shows that Tdrd7 protein co-localized with another RNA binding protein termed Stau1 that is involved in localization of mRNA within specific regions in cells. My research goal was to understand the regulatory function of Tdrd7 in the lens. Specifically, I investigated if different RNAs were localized in the apical and the basal regions of differentiating lens fiber cells and if Tdrd7 deficiency affects these mRNA profiles. To achieve this I expanded the Tdrd7 germline knockout mouse mutant colony and collected Tdrd7 mutant and control (Tdrd7+/- mice that do not exhibit cataracts) embryonic lens tissue for analysis. To study the expression of mRNAs in different locations within fiber cells, I undertook standardization of an approach using Laser Capture Microdissection (LCM). LCM on embryonic lens sections from Tdrd7 null mutants and control were used to isolate the apical and the basal fiber cell regions at mouse embryonic stage E12.5. Once the specific tissues were collected, expression of mRNAs was tested by reverse transcriptase combined with polymerase chain reaction (RT-PCR) analysis. In future, microarray or RNA-sequencing based comparative analysis of gene expression profiles of the apical and basal tissues from Tdrd7 null mutant and control will allow a comprehensive identification of majority of transcripts that are misregulated in these distinct regions as a result of Tdrd7 mutation.