Insertion of a Pax6 consensus binding site into the alphaA-crystallin promoter acts as a lens epithelial cell enhancer in transgenic mice.
ABSTRACT Although the murine alphaA-crystallin promoter is the most commonly used promoter for achieving transgene expression in the developing lens, this promoter directs transgene expression efficiently only in lens fiber cells. The purpose of the present study was to generate promoters capable of directing transgene expression to the entire lens but not to the corneal epithelium.
Transgenic mice were generated with fragments of the murine alphaA- and alphaB-crystallin promoters, as well as with an alphaA-crystallin promoter engineered with the insertion of a Pax6 consensus binding site driving either human growth hormone (hGH) or Cre recombinase genes. hGH expression was evaluated by in situ hybridization and immunohistochemistry. Cre expression was revealed by x-gal staining after crossing Cre transgenic mice with a Cre reporter strain.
Within the lens, the -214/+38 alphaB-crystallin promoter fragment directed transgene expression in the lens epithelium, but not in fiber cells. The native -282/+43 alphaA-crystallin promoter drove transgene expression in the lens fiber cells of several independent lines of transgenic mice, but none of these mice demonstrated significant transgene expression in the lens epithelium. In contrast, the insertion of a 32-bp sequence containing a Pax6 consensus binding site into the -282/+43 alphaA-crystallin promoter reproducibly led to transgene expression in the lens epithelium as well as the lens fiber cells.
The inclusion of a Pax6 consensus binding site within the -282/+43 alphaA-crystallin promoter enhances the ability of this promoter to drive transgene expression in the lens epithelium.
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ABSTRACT: Rybp (Ring1 and YY1 binding protein) is a zinc finger protein which interacts with the members of the mammalian polycomb complexes. Previously we have shown that Rybp is critical for early embryogenesis and that haploinsufficiency of Rybp in a subset of embryos causes failure of neural tube closure. Here we investigated the requirement for Rybp in ocular development using four in vivo mouse models which resulted in either the ablation or overexpression of Rybp. Our results demonstrate that loss of a single Rybp allele in conventional knockout mice often resulted in retinal coloboma, an incomplete closure of the optic fissure, characterized by perturbed localization of Pax6 but not of Pax2. In addition, about one half of Rybp-/- <-> Rybp+/+ chimeric embryos also developed retinal colobomas and malformed lenses. Tissue-specific transgenic overexpression of Rybp in the lens resulted in abnormal fiber cell differentiation and severe lens opacification with increased levels of AP-2alpha and Sox2, and reduced levels of betaA4-crystallin gene expression. Ubiquitous transgenic overexpression of Rybp in the entire eye caused abnormal retinal folds, corneal neovascularization, and lens opacification. Additional changes included defects in anterior eye development. These studies establish Rybp as a novel gene that has been associated with coloboma. Other genes linked to coloboma encode various classes of transcription factors such as BCOR, CBP, Chx10, Pax2, Pax6, Six3, Ski, Vax1 and Vax2. We propose that the multiple functions for Rybp in regulating mouse retinal and lens development are mediated by genetic, epigenetic and physical interactions between these genes and proteins.BMC Developmental Biology 02/2007; 7:39. · 2.79 Impact Factor
Article: Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation.[show abstract] [hide abstract]
ABSTRACT: Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β) encodes an adenosine-5'-triphosphate (ATP)-dependent catalytical subunit of the (switch/sucrose nonfermentable) (SWI/SNF) chromatin remodeling complexes. SWI/SNF complexes are recruited to chromatin through multiple mechanisms, including specific DNA-binding factors (for example, heat shock transcription factor 4 (Hsf4) and paired box gene 6 (Pax6)), chromatin structural proteins (for example, high-mobility group A1 (HMGA1)) and/or acetylated core histones. Previous studies have shown that a single amino acid substitution (K798R) in the Brg1 ATPase domain acts via a dominant-negative (dn) mechanism. Genetic studies have demonstrated that Brg1 is an essential gene for early (that is, prior implantation) mouse embryonic development. Brg1 also controls neural stem cell maintenance, terminal differentiation of multiple cell lineages and organs including the T-cells, glial cells and limbs. To examine the roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific αA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (that is, denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in embryonic day 15.5 (E15.5) wild-type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous and Hsf4 homozygous lenses identified multiple genes coregulated by Brg1, Hsf4 and Pax6. DNase IIβ, a key enzyme required for lens fiber cell denucleation, was found to be downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation, for expression of DNase IIβ, for lens fiber cell denucleation and indirectly for retinal development. These studies demonstrate a cell-autonomous role for Brg1 in lens fiber cell terminal differentiation and identified DNase IIβ as a potential direct target of SWI/SNF complexes. Brg1 is directly or indirectly involved in processes that degrade lens fiber cell chromatin. The presence of nuclei and other organelles generates scattered light incompatible with the optical requirements for the lens.Epigenetics & Chromatin 01/2010; 3(1):21. · 4.46 Impact Factor
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ABSTRACT: Retinal neuron degeneration and survival are often regulated by the same trophic factors that are required for embryonic development and are usually expressed in multiple cell-types. Therefore, the conditional gene targeting approach is necessary to investigate the cell-specific function of widely expressed and developmentally regulated genes in retinal degeneration. The discussion in this review will be focused on the use of Cre/lox-based conditional gene targeting approach in mechanistic studies for retinal degeneration. In addition to the basic experimental designs, this article addresses various factors influencing the outcomes of conditional gene targeting studies, limitations of current technologies, availability of Cre-drive lines for various retinal cells, and issues related to the generation of Cre-expressing mice. Finally, this review will update the current status on the use of Cre/lox-based gene targeting approach in mechanistic studies for retinal degeneration, which includes rod photoreceptor survival under photo-oxidative stress and protein trafficking in photoreceptors.Journal of Ophthalmology 01/2011; 2011:806783.