Generation of NSE-MerCreMer Transgenic Mice with Tamoxifen Inducible Cre Activity in Neurons

To establish a genetic tool for conditional deletion or expression of gene in neurons in a temporally controlled manner, we generated a transgenic mouse (NSE-MerCreMer), which expressed a tamoxifen inducible type of Cre recombinase specifically in neurons. The tamoxifen inducible Cre recombinase (MerCreMer) is a fusion protein containing Cre recombinase with two modified estrogen receptor ligand binding domains at both ends, and is driven by the neural-specific rat neural specific enolase (NSE) promoter. A total of two transgenic lines were established, and expression of MerCreMer in neurons of the central and enteric nervous systems was confirmed. Transcript of MerCreMer was detected in several non-neural tissues such as heart, liver, and kidney in these lines. In the background of the Cre reporter mouse strain Rosa26R, Cre recombinase activity was inducible in neurons of adult NSE-MerCreMer mice treated with tamoxifen by intragastric gavage, but not in those fed with corn oil only. We conclude that NSE-MerCreMer lines will be useful for studying gene functions in neurons for the conditions that Cre-mediated recombination resulting in embryonic lethality, which precludes investigation of gene functions in neurons through later stages of development and in adult

HOXB5 binds to multi-species conserved sequence (MCS+9.7) of RET gene and regulates RET expression

RET gene is crucial for the development of enteric nervous system, and dys-regulation of RET expression causes Hirschsprung disease. HOXB5 regulates RET transcription, and perturbations in transcriptional regulation by HOXB5 caused reduced RET expression and defective enteric nervous system development in mice. The mechanisms by which HOXB5 regulate RET transcription are unclear. Thus, unraveling the regulatory mechanisms of HOXB5 on RET transcription could lead to a better understanding of the etiology of Hirschsprung disease. In this study, we identified and confirmed HOXB5 binding to the multi-species conserved sequence (MCS+9.7) in the first intron of the RET gene. We developed a RET mini-gene reporter system, and showed that MCS+9.7 enhanced HOXB5 trans-activation from RET promoter in human neuroblastoma SK-N-SH cells and in chick embryos. The deletion of HOXB5 binding site interfered with HOXB5 trans-activation. Furthermore, transfection of HOXB5 induced endogenous RET transcription, enhanced the co-precipitation of TATA-box binding protein with the transcription start site of RET, and induced histone H3K4 trimethylation in chromatin regions upstream and downstream of RET transcription start site. In conclusion, (i) HOXB5 physically interacted with MCS+9.7 and enhanced RET transcription, (ii) HOXB5 altered chromatin conformation and histone modification of RET locus, which could facilitate the formation of transcription complex, and enhance RET transcription, (iii) expression of RET was mediated by a complex regulatory network of transcription factors functioning in a synergistic, additive and/or independent manners. Hence, dys-regulation of RET expression by HOXB5 could result in insufficient RET expression and Hirschsprung disease. © 2014 Elsevier Ltd.Link_to_subscribed_fulltex

Plasma exchange in the treatment of early recurrent focal glomerulosclerosis after renal transplantation

We report a 29-year-old female with recurrent focal glomerulosclerosis (FGS) presenting as heavy proteinuria immediately following renal transplantation. From day 13 after transplantation, daily plasma exchange was performed 6 times. Proteinuria decreased from 13 to 0.2 g/day after plasma exchange and remained to be less than 0.1 g/day even 20 months thereafter. Our review of the literature on the use of plasma exchange in treating recurrent FGS suggests this procedure is not useful in patients whose allograft biopsy showed advanced glomerular sclerotic lesions. We propose early plasma exchange may be a therapeutic option in those patients who have no sclerotic lesions in their allograft biopsy. © 1993 S. Karger AG, Basel.Link_to_subscribed_fulltex

Statistics of the generation of <i>NSE-MerCreMer</i> transgenic mice.

a<p>, Number of transgenic lines showing expression of transgene in brain and intestine as detected by RT-PCR or Western blot (WB).</p

Tamoxifen induction of <i>β</i>-galactosidase in neurons of the central nervous system.

<p>Coronal sections of the forebrain (A), rostral (B) and caudal (C) midbrain, and cerebellum (D) of <i>NSE-MerCreMer</i>/<i>R26R</i> double transgenic mice of lines #778 and #805 fed with tamoxifen (TM) or corn oil (CO) and <i>Rosa26R</i> (<i>R26R</i>) mice were analyzed by co-immunofluorescence staining for <i>β</i>-galactosidase (green) and NeuN (red). Superimposed photos of immuno-fluorescence for <i>β</i>-galactosidase (green) and NeuN (red) showed co-localization of <i>β</i>-galactosidase and NeuN immuno-reactivity in neurons in tamoxifen fed <i>NSE-MerCreMer</i>/<i>R26R</i> mice. Arrowheads indicated the Purkinje cells in the cortex of the cerebellum (D). Arrows indicated that few neurons in the corn oil treated forebrain cerebral cortex expressed weak <i>β</i>-galactosidase (A). Dotted square indicated the plans of the coronal sections being analyzed from different regions of the brain. The respective locations of the superimposed photos being taken from the sections were indicated by letters on the drawing of the sections. At least two <i>NSE-MerCreMer</i>/<i>R26R</i> mice from each line were analyzed for each treatment groups.</p

Tamoxifen induction of Cre activity in neurons of the enteric nervous system.

<p>Intestine of <i>NSE-MerCreMer</i>/<i>R26R</i> double transgenic mice of lines #778 and #805 fed with tamoxifen (A, C) or corn oil (B, D) were analyzed by immuno-fluorescence for <i>β</i>-galactosidase (green). Dotted regions were magnified and shown as insets. Abbreviations: m, mucosa; cm, circular muscle; lm, longitudinal muscle.</p

Generation of <i>NSE-MerCreMer</i> transgenic lines.

<p>A, Transgenic construct <i>NSE-MerCreMer</i> consists of a 1.8 kb rat <i>NSE</i> gene promoter and the cDNA encoding the tamoxifen inducible Cre recombinase (MerCreMer). X-gal staining of <i>NSE-MerCreMer</i> and <i>pCAG-CAT-LacZ</i> co-transfected HeLa cells with (+OHT) or without (−OHT) the addition of synthetic ligand 4-OHT. B, PCR amplification of <i>NSE-MerCreMer</i> transgene using <i>CreR1</i> and <i>CreF1</i> primer pair generated a 374 bp DNA fragment from genomic DNA of <i>NSE-MerCreMer</i> transgenic mice (Tg) but not from non-transgenic mice (NTg). ‘+ve’ denotes PCR amplification using the transgenic construct as template DNA. C, RT-PCR analysis showed expression of the transgene (<i>MerCreMer</i>) in the brain (Br, +RT) and intestine (I, +RT) of transgenic mouse. RT-PCR for mouse <i>β</i>-actin (<i>β</i>-<i>actin</i>) was included to check the integrity of the RNA. Reverse transcriptase was omitted in the first strand cDNA synthesis which served as a negative control (−RT) to ascertain the PCR product was not amplified from genomic DNA. D, Western blot analysis using anti-body against ligand binding domain of estrogen receptor α (ERα) detected a protein band of MerCreMer (113 kDa) in the intestine (I), and a protein band of ERα (67 kDa) in the uterus (U) of transgenic mouse. Abbreviation: M, DNA size marker.</p