Unusual topography of bovine rhodopsin promoter-IacZ fusion gene expression in transgenic mouse retinas

The molecular basis of cone photoreceptor-specific gene expression is largely unknown. In this study, we define cis-acting DNA sequences that control the cell type-specific expression of the zebrafish UV cone pigment gene by transient expression of green fluorescent protein transgenes following their injection into zebrafish embryos. These experiments show that 4.8 kb of 5 -flanking sequences from the zebrafish UV pigment gene direct expression specifically to UV cones and that this activity requires both distal and proximal sequences. In addition, we demonstrate that a proximal region located between ؊215 and ؊110 bp (with respect to the initiator methionine codon) can function in the context of a zebrafish rhodopsin promotor to convert its specificity from rod-only expression to rod and UV cone expression. These experiments demonstrate the power of transient transgenesis in zebrafish to efficiently define cis-acting regulatory sequences in an intact vertebrate.

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“…Variegated expression is also commonly observed in mice carrying visual pigment transgenes (17)(18)(19).…”

Section: Resultsmentioning

confidence: 99%

Proximal and Distal Sequences Control UV Cone Pigment Gene Expression in Transgenic Zebrafish

Luo

Williams

Smallwood

et al. 2004

Journal of Biological Chemistry

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“…To restrict transgene expression to specified cell types in the retina, several retinal cell-type-specific promoters were characterized. Regulatory sequences for rhodopsin (12), calcium binding protein 5 (Cabp5), and cellular retinaldehyde binding protein (Cralbp), were previously reported (6). In addition to these promoters, promoter regions of Nrl [expressed in rods (13) (19)] were characterized for this study.…”

Section: Resultsmentioning

confidence: 99%

Controlled expression of transgenes introduced by in vivo electroporation

Matsuda

Cepko

2007

Proc. Natl. Acad. Sci. U.S.A.

612

601

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In vivo electroporation is a powerful technique for the introduction of genes into organisms. Temporal and spatial regulation of expression of introduced genes, or of RNAi, would further enhance the utility of this method. Here we demonstrate conditional regulation of gene expression from electroporated plasmids in the postnatal rat retina and the embryonic mouse brain. For temporal regulation, Cre/loxP-mediated inducible expression vectors were used in combination with a vector expressing a conditionally active form of Cre recombinase, which is activated by 4-hydroxytamoxifen. Onset of gene expression was regulated by the timing of 4-hydroxytamoxifen administration. For spatial regulation, transgenes were expressed by using promoters specific for rod photoreceptors, bipolar cells, amacrine cells, Mü ller glia or progenitor cells. Combinations of these constructs will facilitate a variety of experiments, including cell-typespecific gene misexpression, conditional RNAi, and fate mapping of progenitor and precursor cells.G ain-of-function and loss-of-function studies using transgenic animals have greatly advanced our understanding of the molecular and cellular mechanisms of development and disease. In particular, conditional gene activation and inactivation have been powerful methods for studies of gene function and for the labeling and manipulation of specific cell populations in vivo (1, 2). Site-specific recombination systems (Cre/loxP and Flp/FRT) are widely used to control gene expression in transgenic mice. By crossing two transgenic lines, one expressing Cre (or Flp) under tissue-specific and/or inducible control and the other carrying two loxP (or FRT) sites, DNA recombination can lead to inducible gene expression or loss of gene expression in restricted tissues at specific times (3). Although such strategies are very useful, they are time-consuming and cannot be applied to species that are difficult to manipulate genetically.In vivo electroporation is a convenient technique for the introduction of genes into a variety of animals, including mouse, rat, and chick (4, 5). We previously reported that plasmid DNAs can be easily delivered to developing mouse/rat retinas by in vivo electroporation (6). The plasmid DNAs, electroporated from the scleral side of the postnatal day 0 (P0) retina, are preferentially introduced into retinal progenitor/precursor cells, which give rise to four different cell types; rod photoreceptors in the outer nuclear layer (ONL), bipolar and amacrine cells in the inner nuclear layer (INL), and Müller glial cells, which extend radial processes spanning the entire thickness of the retina [see supporting information (SI) Fig. 7]. The efficiency of electroporation into the developing postnatal retina is quite good, and transgene expression persists at least for a few months. Moreover, compared with other gene transfer methods, such as viral vectors, in vivo electroporation has several advantages. First, various types of DNA constructs, including RNAi vectors, are readily introduced to th...

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“…17 The plasmid also contained an intron and a polyadenylic acid addition site derived from the mouse protamine gene and a eukaryotic consensus ribosomal binding site. After transformation, a clone with correct orientation was selected.…”

Section: Generation Of Transgenic Micementioning

confidence: 99%

“…The fusion gene was purified, and transgenic mice were generated by established techniques as previously described. 17 Mice were screened for the presence of the transgene by either Southern blot analysis or polymerase chain reaction (PCR) of tail DNA. 17 Tail pieces were digested overnight at 55°C in 50 mmol/L Tris (pH 7.5), 100 mmol/L ethylenediaminetetraacetic acid, 400 mmol/L NaCl, 0.5% sodium dodecyl sulfate (SDS) containing 0.6 g/ l proteinase K. PCR was done at 58°C, with primers that amplify 580 bp of transgene-specific sequence P1 (5Ј-GTCCAGCCGGAGCCCCGTG-3Ј) and P2 (5Ј-TGGCACTTGACACTGCTCGTGTTG-3Ј; Figure 1).…”

Section: Generation Of Transgenic Micementioning

confidence: 99%

Platelet-Derived Growth Factor-A-Induced Retinal Gliosis Protects against Ischemic Retinopathy

Yamada

Andõ

et al. 2000

The American Journal of Pathology

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Unusual topography of bovine rhodopsin promoter-IacZ fusion gene expression in transgenic mouse retinas

Cited by 178 publications

References 38 publications

Proximal and Distal Sequences Control UV Cone Pigment Gene Expression in Transgenic Zebrafish

Proximal and Distal Sequences Control UV Cone Pigment Gene Expression in Transgenic Zebrafish

Controlled expression of transgenes introduced by in vivo electroporation

Platelet-Derived Growth Factor-A-Induced Retinal Gliosis Protects against Ischemic Retinopathy

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