Page 1 of Articles 36 in PresS. Physiol Genomics (August 29, 2006). doi:10.1152/physiolgenomics.00092.2006
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BAC Transgenic Mice Express Enhanced Green Fluorescent Protein in Central and Peripheral Cholinergic Neurons
Yvonne N. Tallini1&, Bo Shui1&, Kai Su Greene1, Ke-Yu Deng1, Robert Doran1, Patricia J. Fisher1, Warren Zipfel2, and Michael I. Kotlikoff1*
1
Biomedical Science Department, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 USA 2 School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853 USA
&
These authors contributed equally to the manuscript
Running Title: Targeting Peripheral Cholinergic Neurons
*
To whom correspondence should be addressed:
[email protected] Michael Kotlikoff Professor and Chair Department of Biomedical Sciences Cornell University T4018 VRT, Box 11 Ithaca, NY 14853-6401
Copyright © 2006 by the American Physiological Society.
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2 Abstract The peripheral nervous system has complex and intricate ramifications throughout many target organ systems. To date this system has not been effectively labeled by genetic markers, due largely to inadequate transcriptional specification by minimum promoter constructs. Here we describe transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of endogenous choline acetyltransferase (ChAT) transcriptional regulatory elements, by knockin of eGFP within a bacterial artificial chromosome (BAC) spanning the ChAT locus and expression of this construct as a transgene. eGFP is expressed in ChATBAC-eGFP mice in central and peripheral cholinergic neurons, including cell bodies and processes of the somatic motor, somatic sensory, and parasympathetic nervous system in gastrointestinal, respiratory, urogenital, cardiovascular, and other peripheral organ systems. Individual epithelial cells and a subset of lymphocytes within the gastrointestinal and airway mucosa are also labeled, indicating genetic evidence of acetylcholine biosynthesis. Central and peripheral neurons were observed as early as 10.5 days post coitus in the developing mouse embryo. ChATBAC-eGFP mice allow excellent visualization of all cholinergic elements of the peripheral nervous system, including the submucosal enteric plexus, preganglionic autonomic nerves, and skeletal, cardiac, and smooth muscle neuromuscular junctions. These mice should be useful for in vivo studies of cholinergic neurotransmission and neuromuscular coupling. Moreover, this genetic strategy allows the selective expression and conditional inactivation of genes of interest in cholinergic nerves of the central nervous system and peripheral nervous system.
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3 Keywords: cholinergic, choline acetyltransferase, bacterial artificial chromosome, green fluorescent protein, acetylcholine
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4 Introduction Studies of the development and adaptation of the nervous system have been facilitated by the generation of transgenic mice in which specific neuronal populations express marker proteins such as lacZ or fluorescent proteins (4, 8, 10, 17, 27). The expression of such proteins in sympathetic and parasympathetic nerves would be particularly advantageous for the in vivo study of these widely distributed and poorly understood nervous systems. However, the use of minimal promoter elements derived from parasympathetic or adrenergic genes such as choline acetyltransferase (ChAT) or dopamine
hydroxylase have not provided robust and comprehensive identification of
peripheral nerves when used to direct the expression of transgenes in the mouse (16, 17, 19, 20). The “cholinergic locus” consists of the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) genes, the latter gene entirely contained within the first intron of the former (11, 21). Because ChAT is essential for acetylcholine biosynthesis, several groups have used 5’ flanking DNA from this region to direct transgene expression in selective cholinergic neuron populations (16, 19, 20). While demonstrating expression confined to cholinergic neurons, these constructs do not provide robust peripheral expression. This lack of expression is probably due to the complex nature of ChAT and VAChT regulatory elements, which include enhancer and suppressor elements and significant tissue –specific splicing (14, 18). Thus, for example, Misawa and colleagues recently reported the development of a mouse expressing Cre recombinase and eGFP under control of a 11.3 kb fragment of the cholinergic locus, placing the GFP-IRES-Cre within the VAChT coding region (19); no expression was
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5 observed in parasympathetic nerves in these mice. A bacterial artificial chromosome (BAC) transgenic approach utilizing the ChAT locus to direct expression of eGFP has been described (10). However, marker expression in peripheral cholinergic neurons was not reported in these mice, preventing determination of the effectiveness of the BAC strategy employed. We reasoned that endogenous regulatory elements associated with the ChAT locus should effectively specify transgene expression in central and peripheral neurons, as well as other cells that synthesize acetylcholine, and employed a BAC transgenic approach to preserve these elements. Here we report that transgenic mice using a ChAT locus -spanning BAC, in which eGFP is knocked into exon 3 at the ChAT initiation codon, robustly and selectively express eGFP in all cholinergic nerves of the central and peripheral nervous systems (CNS and PNS), as well as in non-neuronal cells.
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6 Materials and Methods Generation of Tg(RP23-268L19-eGFP)Kot The BAC clone RP23-268L19, containing ~78 kb and ~36 kb of DNA flanking the 5’ and 3’ ends of the ChAT locus, respectively, was obtained from the BAC resource at Children’s Hospital Oakland Research Institute. The eGFP cassette was inserted at the initiation codon of the ChAT coding sequence, which is located within exon 3, by homologous recombination using a targeting vector designed for BAC recombineering (13, 15, 31). ChAT homologous arms were amplified and inserted upstream and downstream of the eGFP sequence in plasmid pBS-eGFP-FRT-Neo/Kan-FRT (Kotlikoff laboratory); ChAT arm1 was a 596 bp fragment upstream of the ATG in exon 3 and was created using forward and reverse primers: 5’-GGC CTT AGA ATA CTT GTG GG-3’ and 5’-CCT AGC GAT TCT TAA TCC A-3’. An XhoI site was added to the 5’ end of the forward primer and a SmaI site was added to the 5’ end of the reverse primer. ChAT arm2 was a 588 bp product downstream of the ATG codon of exon 3 using the forward primer 5’-CCT ATC CTG GAA AAG GTC-3’, with a SpeI site added to the 5’end, and the reverse primer 5’-GAG AAC AAT CAT CCA GAC CA-3’ with a SacI site added to the 5’ end. Plasmid pBS-eGFP-FRT-Neo/Kan-FRT was cut with XhoI and SmaI and ChAT arm1 inserted followed by SpeI and SacI digestion and insertion of ChAT arm2. ChAT(arm1)-eGFP-FRT-Neo/Kan-FRT-ChAT(arm2) cassette was released from the plasmid by XhoI and SacI digestion. The RP23-268L19 BAC was electroporated into the recombinogenic E. coli strain EL250 and positive colonies selected with chloramphenical. EL250 cells carrying RP23-268L19 BAC were electroporated with ChAT(arm1)-eGFP-FRT-Neo/Kan-FRT-ChAT(arm2) and homologous recombined
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7 positive clones selected with kanamycin and chloramphenical. The Neo/Kan cassette was removed by L-arabinose induction of flp recombinase resulting in ChATBAC-eGFP. Following confirmation of homologous recombination by PCR and sequencing the BAC using primers that flanked the upstream of ChAT arm1 to eGFP cassette and downstream of ChAT arm2 to eGFP cassette the ChATBAC-eGFP DNA was prepared using a modified alkaline lysis protocol (QIAGEN kit). Circular intact ChATBAC-eGFP DNA was microinjected into fertilized embryos using standard pronuclear injection techniques. Toe genomic DNA was purified using Puragene (Gentra Systems, Minnesota) and founder mice carrying the BAC transgene were identified by PCR with primer sets RP23P1 5’-ATC CTG GTG TCC CTG TTG -3’ and RP23-P2 5’CGG AAA TCG TCG TGG TAT-3’ in the BAC vector pBACe3.6 yielding a 505 bp product. Founders and offspring were subsequently genotyped with primers to a ChAT-eGFP fragment with a ChATupstream primer 5’-AGT AAG GCT ATG GGA TTC AAT C-3’ and an eGFP-reverse primer 5’-AGT TCA CCT TGA TGC CGT TC-3’, yielding a 620 bp PCR product. Two founder lines were produced that transmitted the transgene in a normal Mendelian inheritance pattern with all offspring appearing grossly normal. Animals were cared for in accordance with guidelines described in the Guide for the Care and Use of Laboratory Animals, using protocols approved by the Cornell IACUC committee. Embryos were removed at specific days post coitus by dissection from the uterus. Pregnant dams and 7 – 18 week old adult mice were euthanized by carbon dioxide inhalation.
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8 Immunohistochemistry Tissues were removed, rinsed in saline, fixed in 10% formalin, dehydrated in graded ethanols, and embedded in paraffin. Paraffin embedded tissues were cut into five micron sections then deparaffinized and rehydrated. Anti-GFP staining has previously been described with the following antibody concentration changes of rabbit anti-eGFP to1:20 (Chemicon International Inc., Temecula, CA) and biotinylated goat anti-rabbit IgG 1:200 (Vector Laboratories, Burlingame, CA) (30). Retrieval of antigen for anti-ChAT staining was performed by steaming in 0.01M Citrate buffer pH 6.0 in a microwave. Sections were blocked with 10% normal rabbit serum with 2X casein for 20 minutes then incubated for 2 hours at 37oC with polyclonal goat anti-ChAT antibody diluted 1:100 in PBS (Chemicon International Inc., Temecula, CA). Biotinylated rabbit anti-goat IgG (Zymed, San Francisco, CA) was applied for 20 minutes at room temperature, followed by PBS washes and incubation for 20 minutes with Streptavidin Peroxidase (Zymed, San Francisco, CA). AEC substrate kit was prepared as directed (Zymed, San Francisco, CA) and applied for 10 minutes. Slides were counterstained with Gill’s #2 Hematoxylin and mounted with Fluoromount G (both from Fisher Scientific Research, Pittsburgh, PA). Controls were included, substituting normal goat serum in place of primary antibody at the appropriate dilution. Immunohistochemistry images were captured either with an Aperio ScanScope (Aperio Technologies, CA) or Olympus DP70 mounted to a LEICA DMLB microscope.
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9 In Vivo and Ex Vivo Imaging In vivo and ex vivo images were obtained using a Lieca MZFLIII stereomicroscope or OV100 macro imaging system (Olympus) with bandpass eGFP filter cubes and fluorescent images captured using a DP70 camera (Olympus). Reconstruction of some images was accomplished using ImageJ (National Institute of Health free software) extended depth of focus plug-in. The acquisition of multiphoton images has previously been described (32).
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10 Results Generation of ChATBAC-eGFP mouse Two ChATBAC-eGFP founder lines, ChAT line 2 and ChAT line 44, were generated by replacing the initial codon of ChAT gene contained in exon 3 with the entire eGFP sequence followed by a polyadenylation signal sequence, thus placing eGFP under control of the endogenous regulatory elements contained in the cholinergic gene locus (Figure 1A) (5). Mice were identified by either a 620 bp or 505 bp PCR product, using primers specific for either the BAC vector or the sequence between ChAT and eGFP (Figure 1A). The offspring appeared grossly normal and the transgene was transmitted in a normal Mendelian inheritance pattern. We systemically evaluated both founder lines for eGFP specificity and expression using fluorescent imaging and immunohistochemistry and no apparent differences were observed between the two founder lines. Following the Mouse and Rat Strain Nomenclature Guidelines the ChATBAC-eGFP mouse was registered with Mouse Genome Informatics as Tg(RP23268L19-eGFP)Kot.
Expression of eGFP in cholinergic nerves of the central nervous system We analyzed the cholinergic CNS regions expressing eGFP and cell specificity of the fluorescence in our mice. Whole brain mounts from adult ChATBAC-eGFP mice express light fluorescence throughout the cerebrum, with intense staining running on the surface of the diencephalon caudally to the mesencephalon (Figure 2A). Highly fluorescent fibers in the pons and medulla were identified as cranial nerves III, V-XII, and cervical spinal nerves (Figure 2A-B). No fluorescence was observed in the
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11 cerebellum, consistent with minimal cholinergic neurotransmission in that region. Immunodetection of eGFP revealed specific neuronal populations expressing the transgene (Figure 2C); fibers and/or cell bodies were specifically labeled in the cortex, caudate putamen, accumbens, medial habenular, basolateral amygdaloid, anterior olfactory, facial, spinal V, medial vestibular, prepositus hypoglossal, solitary tract, and ambiguous nuclei, as well as in the spinal trigeminal tract, consistent with previous findings based on ChAT immunodetection (20, 22). The entire spinal cord was intensely fluorescent and cross-sections at the thoracic and lumbar spinal cord segments showed eGFP positive immunostaining in large cell bodies, as well as in varicose nerve fibers and ventral nerve roots of the ventral horn, consistent with expression in cell bodies and axons of - motor neurons (Figures 2D and E). Prominent eGFP expression was also detected in the intermediolateral cell column (lamina VII) located in the lateral horn of the thoracic and upper lumbar spinal cord, which gives rise to autonomic preganglionic sympathetic neurons. Cell bodies and processes in the dorsal horns (lamina I-VI) of the spinal cord and dorsal root ganglia, representing nerves involved in carrying sensory information from peripheral organs, were also stained (Figure 2E). To document the cholinergic specific expression of eGFP at the cellular level, we performed immunohistochemistry using antibodies against ChAT and eGFP in sequential brain slices. Anti-eGFP immunostaining was more prominent in cell bodies and neuronal processes when compared to anti-ChAT immunostaining (Figure 2F). Comparison of the same regions in serial sections indicated that eGFP and ChAT were expressed in the same neuronal cell populations throughout the brain except in two regions: olfactory cortex and
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12 amygdaloid. To our surprise anti-ChAT did not stain these two areas even though reports by other groups and our ChATBAC-eGFP mice expressed eGFP in these regions; this is most likely attributable to differences in immunohistochemistry techniques (20, 22). In double labeling experiments, eGFP –expressing neurons were co-labeled by antibodies directed against neuronal nitric oxide (nNOS), a neuron specific synthatase, or synaptophysin, an integral membrane protein expressed in the presynaptic vesicle in neurons (not shown). Moreover, an astrocyte –specific antibody directed against glial fibrillary acidic protein, did not label eGFP –positive cells in brain slices (not shown). Taken together these data demonstrate that ChATBAC-eGFP mice express eGFP in cholinergic cell bodies and processes of the CNS, including elements specific to the autonomic nervous system.
Expression of eGFP in cholinergic nerves of the peripheral nervous system We next evaluated eGFP fluorescence in elements of the PNS in both in vivo and ex vivo preparations, to determine the extent to which eGFP is expressed in ChATBACeGFP mice cell bodies and extensive processes of cholinergic nerves, including motor neuron axons and pre and post –synaptic autonomic neurons. As shown in Figure 3A, adult mice display robust eGFP fluorescence on the lateral surface of the jaw in all branches of the facial nerve, which carries motor axons from cell bodies in cranial nerve VII to facial muscles. Fluorescence extended through nerve branches to the neuromuscular junction (Figure 3B), indicating efficient axonal transport of transgene protein and/or mRNA. Superficial motor neurons running along the lateral and medial surfaces of the thoracic and pelvic limbs also expressed eGFP (data not shown).
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13 Similarly, phrenic motor nerves projecting to the diaphragm from cervical nerves 3-5 displayed intense in vivo fluorescence (Figure 3C). These data indicate effective marking of postganglionic motor neurons of the somatic peripheral nervous system. Of perhaps more significance, eGFP was observed in the parasympathetic nervous system, which has not been effectively labeled by transgenic methods (16, 20). As shown in Figure 3C, the vagus nerve trunk displayed clear fluorescence within the thoracic cavity, although the fluorescence intensity of the vagus both in the neck and thoracic cavity was distinctly lower than observed in somatic motor nerves. This likely reflects the smaller diameter preganglionic autonomic axons and lower axon density of the nerves, although lower promoter strength in vagal preganglionic neurons could not be excluded. eGFP expression was also observed in ganglionic neurons; three different ganglia, representing predominately parasympathetic, sympathetic, or mixed neurons (6, 28), were evaluated. The parasympathetic ciliary ganglion located on the dorsal surface of the eye receives preganglionic fibers from the Oculomotor nerve (III) and synapses on postganglionic fibers projecting to smooth muscle of the eye (6); eGFP was expressed in the preganglionic Oculomotor nerve running parallel with the optic nerve, the preganglionic Oculomoter nerve at the ciliary ganglion, and postganglionic cell bodies and ciliary nerve fibers (Figure 3E). The second ganglia we assessed was the paravertebral chain ganglia containing sympathetic preganglionic nerves that exit the ventral root of the thoracic and lumber spine to form synapses on postganglionic cell bodies. As shown in Figure 3F, paravertebral ganglia display eGFP fluorescence in processes running to, and synapsing within, the ganglia. Finally, we evaluated mixed
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14 sympathetic and parasympathetic neurons within the pelvic ganglia located on the dorsal surface of the prostate gland. These ganglia receive inputs from lumbar and sacral regions of the spinal cord and are the major autonomic ganglia supplying postganglionic nerves to reproductive organs, the lower urinary tract, and the lower bowel (28). As shown in Figure 3G, the major nerves of the pelvic ganglia were easily distinguished by eGFP fluorescence in ChATBAC-eGFP mice (28). Postganglionic, parasympathetic process innervating target organs also display strong eGFP fluorescence, an important advantage of ChATBAC-eGFP mice for physiological studies. As shown in Figure 3H, postganglionic fibers were labeled in a variety of target organs, including within the extensive submucosal plexus of the gastrointestinal tract. Single non-neuronal epithelial cells were also labeled within the gastrointestinal mucosa (also see below). The sweat gland is unusual in that sympathetic innervation switches during development from catecholaminergic to cholinergic; Figure 3I demonstrates that eGFP fluorescence is observed in hind foot sweat glands in adult ChATBAC-eGFP mice, demonstrating that this transition has occurred (26). Taken together, these data demonstrate the ability to visualize postganglionic projections of the somatic motor, sympathetic and parasympathetic PNS in vivo and ex vivo.
Cellular Expression of eGFP in the PNS To more carefully examine the cellular pattern and neuronal specific nature of ChAT driven eGFP expression in target tissues we performed immunostaining using antiGFP antibodies. As shown in Figure 4A, eGFP was detected in the cytoplasm of cell bodies present within ganglia and processes of nerves similar to earlier findings (12). To
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15 further document the neuronal specificity of ChATBAC-eGFP mice in the PNS we performed immunohistochemistry using antibodies against eGFP and nNOS in the intestine. Approximately 50% of the cells within the ganglia of Auerbach’s and Meissner’s plexus co-labeled with eGFP and nNOS, a ratio similar to previous reports in the mouse (data not shown) (24). Choline acetyltransferase has also been reported to be present in non-neuronal cells within lung and intestine (29). Prominent eGFP immunostaining was observed in non-neuronal tissues in ChATBAC-eGFP mice (Figure 4B), consistent with the observation of intense focal fluorescence in these tissues (Figure 3H). To identify the cell populations in the lung and intestine expressing eGFP, we used specific markers for epithelial cells (keratin), neuroendocrine cells (synaptophysin), lymphocytes (CD38), macrophages (MAC387), and goblet cells (Ascian blue and Periodic Acid Shift). eGFP colocalized with keratin and a subpopulation of lymphocytes but not with neuroendocrine or goblet cell markers, consistent with the expression of the transgene in epithelial cells and T lymphocytes (data not shown) (29).
Embryonic Development The high degree of fidelity of eGFP expression in cholinergic cells conferred by the BAC transgene provides an effective way to study the development and adaptation of cholinergic neurons, including the targeting of motor neurons to specific muscles and the complex ramifications of the parasympathetic nervous system. To confirm the early embryonic expression of eGFP we examined ChATBAC-eGFP embryos at several stages of development. At all stages of fetal development evaluated ChATBAC-eGFP positive embryos could easily be distinguished from negative littermates (Figure 5). As early as
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16 10.5 dpc, cranial ganglia displayed robust eGFP fluorescence, the eGFP fluorescence between the somites was identified in a cross-section taken below the level of the heart as ventral motor neurons. In addition, sympathetic ganglion and enteric neurons were identified in this cross-section (Figure 5A-C). By 13.5 dpc eGFP fluorescence was prominent between the digits of both fore- and hind limbs (Figure 5D and E). Figure 5F shows a cross-section taken at the brachial level with cholinergic dependent eGFP fluorescence in the bilateral ventral roots, bilateral sympathetic ganglia, left vagal sympathetic trunk and fine projections to the limbs. At this stage fluorescent nerve projections to the gastrointestinal organs were apparent including significant ramifications within the smooth muscle and mucosal layers, which became progressively more extensive by 16.5 dpc and 18.5 dpc, consistent with previous literature (Figure 5G) (7).
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17 Discussion The peripheral nervous system is an extensive, diverse, and highly adaptive system that plays essential roles both as the conduit for central control of higher order function, and as a distributed nervous system capable of complex pattern generation and processing in local ganglia. The extensive ramifications, complex wiring, and diverse expression of neurotransmitters, particularly in the autonomic nervous system, have complicated neurophysiological evaluation of the PNS. Recently, substantial progress has been made in the understanding of complex central processing through the use of genetic approaches that enable the targeting of specific neuronal populations and also provide a mechanism to regulate gene expression in these cells. Here we report specific and effective labeling of the cholinergic nervous system using BAC transgenic methods that exploit the ChAT gene locus. The ChAT gene resides in a complex “cholinergic locus” with numerous poorly understood tissue specific regulatory elements and a second gene, VAChT, contained within the first intron (11, 20). Transgenic mice expressing eGFP under control of minimal ChAT promoter constructs lack cholinergic specificity and parasympathetic neuronal labeling (16, 20), emphasizing the difficulty of reconstituting endogenous gene expression patterns with this approach. More recently, a BAC strategy utilizing the ChAT locus was employed as part of a large scale CNS gene expression atlas (10). This strategy resulted in robust expression of GFP in central neurons, however no evaluation of peripheral expression was reported and therefore neither the degree of expression in the PNS or other cholinergic cells, nor the extent, to which neuronal processes are labeled, is available. We employed a similar strategy in which eGFP cDNA is knocked into the first ChAT coding exon within a large (173 kb)
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18 genomic DNA fragment by homologous recombination, however the BAC targeting approach differed in two significant ways. First, we used homology sequences upstream and downstream of the initiation codon, which flanked the insertion eGFP cassette, and confirmed the insertion site within the BAC by PCR. Second, we removed the recombination selection (NEO) cassette prior to injection of the BAC (13). The high throughput strategy employed by Gong et al (2003) results in the retention of the selection cassette, as well as vector sequences such as the R6K origin of replication within the ChAT locus. The extent to which these differences affected peripheral expression are not clear, however, and in both strategies the retention of extensive 5’ and 3’ flanking sequence and the insertion of the eGFP reporter gene at the ChAT start codon should preserve 5’ untranslated nucleotides associated with the normal transcript. A further advantage of this approach is that the use of this construct as a randomly inserted genetic element (pronuclear injection) enables preservation of the endogenous ChAT alleles, whereas knockin strategies usually require inactivation of the targeted allele. Progenies from both ChATBAC-eGFP lines displayed eGFP expression in a pattern that was qualitatively the same suggesting BAC transgenic mice may have less integration site positional insertion effects and are insulated from regulatory elements of neighboring genes because they harbor the majority, if not all of the chromatin domain insulator elements (25). While the random insertion of large DNA fragments may occasion fragmentation or recombination, lines can be effectively screened by PCR strategies that take advantage of the BAC vector DNA. Using this tactic, random primers to the BAC vector were designed and one such example is shown in Figure 1A or the presence of both BAC vector and insert junction of DNA. While we did not attempt to
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19 quantify the gene copy number in our ChATBAC-eGFP lines, pronuclear injection of BAC transgenes usually results in the integration of only a few copies/genome of the BAC DNA (1, 23). Despite the likely lower copy number of large BAC inserts, eGFP expression was found to be remarkably high, and excellent axonal transport was achieved without fusion of the tau microtubule binding protein (23). We have observed this phenomenon in several BAC transgenics and attribute the strong expression to the fact that the large genomic provides the complete regulatory context required for robust gene expression. Finally, as previously noted, the BAC approach requires little or no knowledge of the underlying gene regulatory elements and is amenable to both relatively high throughput approaches (10). The specific strategy used here should be an effective way to obtain lineage specific transgene expression that extends to cholinergic nerves within the autonomic nervous system, such as the expression of Cre recombinase or specific neural factors. Immunostaining using antibodies directed against eGFP and ChAT indicated extensive co-labeling of central and peripheral cholinergic neurons, indicating that the genetic indicator serves to accurately reflect ChAT expression. The single exception to this overlap was the failure of the anti-ChAT antibody to recognize a small subset of neurons with previously reported ChAT expression using our staining methods, further emphasizing the advantage of a broadly expressed transgene marker. In the intestine eGFP and ChAT colocalized to cell bodies within the ganglia and neuronal processes of Auerbach’s and Meissner’s plexus, demonstrating eGFP -specific expression in cholinergic neurons of peripheral organs. The expression of eGFP in central and peripheral cells was observed at the earliest time point evaluated, 10.5 dpc, with
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20 progressive rostral to caudal innervation of the intestinal tract during embryonic developments. Interestingly, at 13.5 dpc intense fluorescence was observed between the fore- and hind limb digits; these axons may be responsible for maintaining the appropriate synapse to skeletal muscle cell ratio as homozygous knockout ChAT mouse embryos develop wrist drop (3). In contrast to the common role of acetylcholine in neuronal cells of the CNS and PNS its cellular function(s) in non-neuronal cells is limited. Our observation of intense eGFP fluorescence and staining in epithelial cells of airways and alimentary tract and in a subset of lymphocytes is consistent with reports of ChAT mRNA and protein within these cell types (29) and demonstrates the potential use of ChATBAC-eGFP mice as an experimental model to study the non-classic role of acetylcholine under normal and pathological conditions. The availability of a genetic marker of cholinergic activity should be useful for the study of the development of the cholinergic nervous system, as well as genetic and acquired autonomic neuropathies. Moreover, we demonstrate the ability to effectively target this system by genetic means, enabling tissue –specific gene expression and gene inactivation. ChATBAC-eGFP mice should advance our understanding of the in vivo development of the cholinergic nervous system, adaptive changes that occur under pathophysiological conditions, and the in vivo regenerative capabilities of the CNS and PNS after injury.
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21 Acknowledgements We thank Dr. Brian Summers and Dr. Sean McDonough for help with histological analysis, Dr. Drew Noden for embryonic evaluation, Shaun Reining for animal support and Jane Lee for vector construct support. This work was supported by National Institutes of Health Grants HL45239 and DK65992 (to M.I. Kotlikoff) and HL76999 (to Y. Tallini).
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27 FIGURE 1 Generation of ChATBAC-eGFP transgenic mice. A) Diagram of BAC strategy to insert eGFP into the 3rd exon to replace the start codon of the ChAT gene. Medium grey arrow denotes orientation of ChAT gene. Prior to pronuclear injection the neomycin/kanamycin cassette was removed. B) Detection of the transgene by PCR. Lane 1 is a 1 kb DNA ladder, lane 2 shows a 505 bp product from primers located in the BAC vector (light grey arrows in panel A: see Material and Methods RP23-P1 and RP23-P2 primers;) and lane 3 shows a 620 bp product from primers starting in the ChAT gene locus and ending in the eGFP portion of the transgene (black arrows in panel A: see Material and Methods ChAT-upstream and eGFP-reverse primers).
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28 FIGURE 2 eGFP fluorescence is restricted to the central nervous system in ChATBAC-eGFP mice. A) light (left) and fluorescence (right) dissecting scope ex vivo whole brain images from adult ChATBAC-eGFP mice. B) Close-up in vivo view of the ventral surface of the pons, medulla, and rostral spinal cord showing fluorescence of the cranial and spinal nerves. C) Anti-eGFP antibody immunostaining from rostral to caudal cross-sections of the brain. D) Ventral view of spinal cord from postnatal day two mouse E) Anti-eGFP immunostaining in the thoracic and lumbar segments of the spinal cord. Note the labeling of the ventral nerve root exiting the spinal cord and dorsal root ganglia. F) antiChAT (left) and anti-eGFP (right) serial sections of the medial habenular nucleus of the brain shows the same cell bodies and nerve processes possess eGFP and ChAT. Abbreviations: III, Oculomotor; V, Trigeminal; VI, Abducens; VII, Facial; IX, Glossopharyngeal; X, Vagus; XI, Accessary; XI, Hypoglossal; AO, Anterior Olfactory Nucleus; CPu, Caudate putamen; Acbc, Accumbens Nucleus Core; AcbSh, Accumbens Nucleus Shell; OC, Olfactory Cortex; LGP, Lateral Globus pallidus; Mnb, Medial Habenular Nucleus; BL, Basolateral Amygdaloid Nucleus; 7, Facial Nucleus; Sp5, Spinal 5 Nucleus; MVeMC, Medial Vestibular Nucleus; Pr, Prepositus Hypoglossal Nucleus; 7PA, Facial Nucleus Proximal Axons; 12,Hypoglossal nucleus; Sol, Solitary Tract Nucleus; Amb, Ambiguus Nucleus; VH, ventral horn; L9, lamina 9; DH, dorsal horn; VNR, ventral nerve root; DRG, dorsal root ganglia. Scale Bars: A, 1 mm; B, cranial left 1 mm; cranial right 500 µm; cervical nerves 1mm; C, 500 µm; D, 1mm; E, 250 µm; and F, 50 µm.
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29 FIGURE 3 In vivo and ex vivo eGFP fluorescence in the peripheral nervous system in ChATBACeGFP mice A) Light (left) fluorescence (right) of the lateral surface of the jaw shows fluorescence is confined to the dorsal buccal branch (*), ventral buccal branch (**) and marginal mandibular branch (***) of the facial nerve. B) Two photon image of a synapse on a single skeletal muscle fiber. C) Light (left) fluorescence (right) of the thoracic cavity exposing the right and left phrenic nerve (arrows) coursing to the diaphragm and the right vagus nerve on the esophagus (*) D) blow up of the box in C showing the projections of the phrenic nerve on the diaphragm. E) Ciliary ganglia located on the dorsal surface of the eye. Double arrows pointing to the optic nerve and the single arrow is oculomotor nerve running adjust. The ciliary ganglia is denoted by the star. Note the process leaving the ciliary ganglia. F) Two paravertebral fluorescent chain ganglia attached by ganglionic nerve fibers. G) Highly fluorescent pelvic ganglia showing accessory, hypogastric, and pelvic nerve H) Nerve fibers and punctuate labeling in target organs. Arrows denote nerves and punctuate labeling of interest. I) eGFP labeled nerve fibers in the hind footpad sweat gland. Abbreviations: A, accessory nerve; H, hypogastric nerve; PL, pelvic nerve. Scale bar: A, 500 µm; C, 1mm; E-G, 500 µm, H, 100 µm; I, 25 µm.
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30 FIGURE 4. Cellular expression of eGFP staining in ChATBAC-eGFP mice. A) eGFP expression in ganglia and nerve processes in peripheral target organs. In the intestine note labeling in the Auerbach’s and Meissner’s plexus B) eGFP expression in non-neuronal tissues including epithelial cells in the lung and villi of the intestine and a subset of lymphocytes in Peyer’s patch of the intestine. Scale bar: 50 µm.
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31 FIGURE 5 Embryonic expression of eGFP in ChATBAC-eGFP mice. A) Light (left) fluorescence (right) of a 10.5 dpc embryo. B) Enlarged view of boxed area in A showing cranial and spinal nerves (indicated by A). C) Cross-section showing ventral motor neuron (indicated by B), single arrow pointing to sympathetic ganglion and double arrow to enteric neurons. D) Light (left) fluorescence (right) of a 13.5 dpc embryo. E) Enlarged view of boxed area in C showing fluorescence in the forelimb. F) Cross-section taken at the brachial level of a 13.5 dpc embryo. Ventral motor neuron (*), single arrow pointing to sympathetic ganglion and double arrow to left vagal sympathetic trunk. G) Organs from a 13.5 dpc embryo showing cholinergic projections in the stomach and intestine. Abbreviation: X, Ganglia Vagus Scale bar: A, D and F, 1 mm; B and E, 250 µm; C, 100 µm; and G, 50 µm.
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Figure 1
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Figure 5
