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

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 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

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

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

Tamoxifen induction of <i>β</i>-galactosidase in the central nervous system and the intestine of <i>NSE-MerCreMer</i>/<i>R26R</i> mice.

<p>Western blot analysis of protein extracted from the brain (Br), spinal cord (SC) and small intestine (SI) of <i>NSE-MerCreMer</i>/<i>R26R</i> mice treated with tamoxifen (TM) or corn oil (CO); <i>Rosa26R</i> mice (<i>R26R</i>) and <i>Z/EG</i> mice. Except for the small intestine of <i>Z/EG</i> mice that 20 µg of protein was loaded, 100 µg of protein was analyzed for all the other samples.</p

Spatial and temporal expression of transgene in <i>NSE-MerCreMer</i> mice.

<p>Total RNA was isolated from neural and non-neural tissues from postnatal week-4 transgenic mice (A) or brain and intestine of transgenic embryos of various embryonic stages (B) from lines #778 and #805. Expression of the transgene (<i>NSE-MerCreMer</i>) was analyzed by RT-PCR. RT-PCR for mouse <i>β</i>-actin (<i>β</i>-<i>actin</i>) was included to check the integrity of the isolated 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. Abbreviations: Br, brain; I, intestine; H, heart; Li, liver; Sp, spleen; Lu, lung; Ki, kidney.</p

Localization of MerCreMer protein to the enteric neurons in the intestine of <i>NSE-MerCreMer</i> transgenic mice.

<p>Immuno-reactivity for Tuj1 (green; neuronal marker) was localized to the enteric ganglion plexus at the myenteric region between the circular muscle and the longitudinal muscle of the intestine of non-transgenic mice (C), and transgenic mice (D). In contrast, immuno-reactivity for ERα (red) was only detected at the myenteric plexus in the intestine of transgenic mice (F), but not in non-transgenic mice (E). Superimposed photos of immuno-fluorescence for ERα (red) and Tuj1 (green) of transgenic intestine (H), and non-transgenic intestine (G) showed co-localization of ERα and Tuj1 immuno-reactivity in transgenic intestine. Dotted regions are magnified and showed as insets. Abbreviations: m, mucosa; cm, circular muscle; lm, longitudinal muscle.</p

Schematic diagram of transgenic mice crossing and tamoxifen induction of Cre activity.

<p>MerCreMer protein in the cytoplasm of the neurons of <i>NSE-MerCreMer/R26R</i> mice moves into the nucleus after binding to tamoxifen, mediates the excision of the DNA stuffer. The <i>LacZ</i> gene is transcribed. <i>NSE-MerCreMer</i> mice were crossed to <i>Rosa26R</i> reporter mice (<i>R26R</i>) to generate <i>NSE-MerCreMer/R26R</i> double transgenic mice. Tamoxifen or corn oil was administered to postnatal week-4 double transgenic mice at day 1, 2, 3 and 4. Brain and intestine were harvested at day 7, and processed for Western blot analysis and immuno-fluorescence staining for <i>β</i>-galactosidase.</p