Browsing by Subject "Development"
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Item LENS SPECIFIC RLIP76 TRANSGENIC MICE DEVELOP MICROPHTHALMIA: A NOVEL ANIMAL MODEL TO STUDY LENS DEVELOPMENT(2013-04-12) Sahu, MukeshPurpose: The direct role of RLIP76 in eye development has not been investigated yet. In order to elucidate the role of RLIP76 in lens development, we have engineered lens specific RLIP76 Transgenic (RLIP76 Tg) mice and investigated the effect of RLIP76 overexpression on lens development. Methods: Lens specific RLIP76 Tg mice were generated in collaboration with Dr.Wakamia Maki. Mouse RLIP76 cDNA, 1885 base pairs long, was cloned in δenɑA promoter containing vector. Five founders were identified by southern blot analysis, and bred with C57BL/6J mice. Genotyping was performed to confirm the identity of offspring's by polymerase chain reaction using genomic DNA purified from mouse tail. Lens tissue organization of RLIP76 Tg and wild type mice was compared by Hematoxylin & eosin (H&E) staining. DNA microarray was used to compare the gene expression profiles of lens in RLIP76 Tg and wild type mice. The expression levels of various transcription factors such as Pax6 HSF1 and HSF4b was also determined using western blot and immunohistochemistry. Results: The most striking result from these studies was that the eyes of RLIP76 Tg mice were smaller in size as compared to those of wild type. In RLIP76 Tg mice the weight of the eyes was smaller and the lens development was impaired. RLIP76 Tg mice exhibited disruption in lens tissues organization and the expression of a multitude of genes was affected in lens of these mice. The expression of transcription factors Pax6, HSF1 and HSF4b, involved in eye development was suppressed in RLIP76 Tg mice. Actin cytoskeleton organization in RLIP76 Tg lens was severely disrupted. Cdc42, a regulator of actin cytoskeleton organization, activation level was reduced in RLIP76 Tg lens compared to wild type lens. Conclusions: The eyes of RLIP76 Tg exhibit features of microphthalmia and show poor lens development. These mice provide a good animal model for elucidating the mechanisms involved in lens development. Our results suggest that the lens developmental abnormalities in RLIP76 Tg mice may be due to the suppression of Pax6, HSF1, HSF4b and HSP's expression resulting in actin cytoskeleton disorganization.Item The Development and Evaluation of an Oligonucleotide Probe for Use in Quantifying Human Mitochondrial DNA Using the Quantiblot Human DNA Identification Kit(2003-07-01) Cooksley, Joseph David; John Planz; Joseph Warren; Arthur EisenbergCooksley, Joseph David, The Development and Evaluation of an Oligonucleotide Probe for Use in Quantifying Human Mitochondrial DNA Using the Quantiblot Human DNA Identification Kit. Master of Science (Forensic Genetics), August 2003, 93pp., 3 tables, 14 figures, 1 appendix, references, 17 titles. Nucleotide sequences from coding regions in the mitochondrial DNA genome were evaluated for use in designing an oligonucleotide probe specific to humans that could be used with Quantiblot Human DNA Identification kit from Applied Biosystems. Two probes were designed and evaluated using the conditions, reagents, and protocols recommended in the kit instructions, as well as with less stringent conditions than those recommended in the kits instructions. Probe 1 bound to amplified mtDNA at low stringency conditions, with greater probe amount, with heat denaturation of the probe, and more HRP-SA enzyme conjugate added than recommended in the protocol. Probe 1 did not hybridize to mtDNA extracted from a buccal swab, bloodstain cards, or a whole blood sample. Probe 2 did not show any hybridization to mtDNA when evaluated.