ARTICLE |
Correspondence to: Gábor Szabó, Lab. of Molecular Neurogenetics, Inst. of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, PO Box 521, Temesvári krt. 62, Szeged, Hungary.
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Summary |
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In the olfactory bulb (OB) of a transgenic mouse line that carries the bacterial LacZ gene under the control of the 5'-regulatory region of the GAD67 gene, expression of the ß-galactosidase was confined almost exclusively to the non-GABAergic mitral and tufted cells. By light microscopy, enzyme histochemistry showed strong staining in the cell bodies and faint diffuse staining in the axons and dendrites. With immunohistochemistry for ß-galactosidase the entire cytoplasm, including the axons and dendrites, was strongly stained. By electron microscopy, ß-galactosidase enzyme histochemistry resulted in a submicroscopic reaction product that was diffusely distributed in the cytoplasm of neurons. In addition, large deposits of the reaction product were also seen attached to the cytoplasmic side of the membranes. In contrast, when the intracellular localization of ß-galactosidase was determined by immunohistochemistry, homogeneous cytoplasmic staining was obtained that filled the entire cytoplasm including the terminal dendrites and fine axons. Therefore, synaptic contacts of the ß-galactosidase-positive output neurons with other ß-galactosidase-negative neuronal cells were readily recognized in the OB. As we demonstrated, transgenic mouse lines expressing the LacZ reporter gene in a well-defined neuronal subpopulation can be used to follow ß-galactosidase-positive neurons and to directly identify their synaptic connections. (J Histochem Cytochem 45:1147-1155, 1997)
Key Words: olfactory system, output neurons, neuronal labeling, GAD-LacZ fusion protein, transgenic expression
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Introduction |
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Throughout adult life, ongoing neurogenesis and synaptic remodeling take place in the olfactory system due to continuous replacement of the olfactory receptor neurons (
Transgenic mouse lines expressing the LacZ reporter gene in a well-defined neuronal population have already been used to explore the mechanisms involved in cortical development (
Targeting of bacterial ß-galactosidase as a marker for CNS neurons can be achieved by using promoters of neuron-specific genes such as neuron-specific enolase (
Some of the transgenic mouse lines express ß-galactosidase in the OB (
The aim of our study was to find a reliable method to identify synaptic connections of a transgenically labeled neuronal population. For this purpose, here we characterize the intracellular distribution of the transgene product in the output neurons within the OB of a transgenic mouse line Tg(GAD67lacZ7.5)1 (Katarova et al., submitted for publication) by using enzyme and immunohistochemical detection of ß-galactosidase at the light and electron microscopic levels.
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Materials and Methods |
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Transgenic Mice
A number of transgenic mouse lines were generated that carry the bacterial ß-galactosidase marker gene attached to a segment of the mouse gene encoding the 67-kD isoform of the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD67) (Katarova et al., submitted for publication). The constructs included the first intron and exon, as well as progressively longer portions of the 5'-flanking region of the regulatory DNA region of the GAD67 gene (Katarova et al., submitted for publication). In this study, one of the transgenic mouse lines with 5 KB of the 5'-flanking region, Tg(GAD67lacZ7.5)1, was selected to study the ß-galactosidase expression pattern within the OB. Animals were maintained and handled according to approved institutional guidelines.
Histological Processing
Two-month-old heterozygous transgenic males (n = 4) were deeply anaesthetized and transcardially perfused with PBS (137 mM sodium chloride, 2.7 mM potassium chloride, 8 mM sodium phosphate dibasic, and 1.47 mM potassium phosphate monobasic), followed by a mixture of 2% para-formaldehyde and 0.25% glutaraldehyde in 0.1 M PIPES buffer (pH 6.9). Brains were removed and postfixed for 1 hr in the same fixative. For light microscopic observation, 15-30-µm-thick sections were cut on a cryostat after cryoprotection in 20% sucrose with 0.02% sodium azide in PBS. For electron microscopy, 100-µm-thick vibratome sections were prepared. Until further use, all the sections were kept in PBS at 4C. Half of both kinds of sections were processed for enzyme histochemistry, and the rest of the sections were prepared for ß-galactosidase immunohistochemistry. Alternate sections processed by enzyme histochemistry were counterstained with 1% neutral red. For routine histology, all stained cryostat sections were dehydrated in an ascending series of ethanol, cleared in xylol, and mounted in Entellan (Merck; Darmstadt, Germany).
ß-Galactosidase Enzyme Histochemistry
Enzyme histochemical detection of ß-galactosidase was performed by incubating the sections in PBS containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM magnesium chloride, 0.02% Nonidet P-40 (w/v), 0.01% sodium deoxycholate (w/v) (detergents were omitted during processing for electron microscopy), and either 1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) (Boehringer Mannheim; Vienna, Austria) or 1 mg/ml Bluo-gal (5-bromo-3-indolyl-ß-D-galactoside) (BRL Life Technologies; Gaithersburg, MD) as substrate for 3-5 hr at 37C. Sections from non-transgenic animals were used as negative controls. X-gal, which yields a brighter blue reaction product than Bluo-gal, was used for light microscopic observation. Bluo-gal was applied for electron microscopy because it produces a heavily electron-dense precipitate and is less diffusable than X-gal (
ß-Galactosidase Immunohistochemistry
For ß-galactosidase immunohistochemistry, sections were kept for 30 min in 0.6% H2O2 in 10% methanol to block endogenous peroxidase activity. After brief rinsing, nonspecific antibody binding was suppressed by normal goat serum diluted 1:20 in Tris-buffered saline (TBS; 10 mM Tris-HCl, pH 7.6, 150 mM NaCl) with 1% bovine serum albumine (BSA) for 1 hr. Then sections were incubated with monoclonal anti-ß-galactosidase antibody (Promega; Madison, WI) diluted 1:1500 in TBS with 1% BSA for 1 hr at room temperature (RT). After rinsing in TBS the sections were incubated with biotinylated sheep anti-mouse IgG F(ab') (Amersham; Poole, UK) diluted 1:2000 in TBS with 1% BSA for 1 hr at RT. After several rinsings, the bound antibody was visualized by the avidin-biotin-peroxidase procedure using the Vectastain ABC Elite kit (Vector Laboratories; Burlingame, CA) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) as chromogen.
Control sections incubated without the primary antibody, as well as sections from non-transgenic animals, showed very low background and no specific cell staining.
Electron Microscopy
For electron microscopy, the vibratome sections from transgenic and control animals stained with anti-ß-galactosidase were postfixed in a mixture of 2% paraformaldehyde and 2% glutaraldehyde in PBS. Free aldehydes were carefully removed by several changes of PBS and in freshly prepared 1% sodium borohydride in PBS for 30 min. The tissue was postfixed for 1 hr in 1% OsO4 (Sigma; St Louis, MO) in PBS, washed in PBS, dehydrated in ethanol (30%, 50%, 70%), stained with 1% uranyl acetate in 70% ethanol for 1 hr, dehydrated in ethanol (80%, 90%, 100%), infiltrated, and embedded in Durcupan (Fluka; Buchs, Switzerland). Ultrathin sections were cut and stained with uranyl acetate and lead citrate. The sections were observed in Tesla Bs500 and Zeiss EM-10C electron microscopes. In the control sections, we did not observe any electron-dense precipitate.
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Results |
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To study the transcriptional regulation of the mouse GAD67 gene, we have constructed several transgenic mouse lines carrying the bacterial LacZ gene under the control of progressively longer portions of the 5'- upstream regulatory region of the GAD67 gene (Katarova et al., submitted for publication). Preliminary results indicated a striking but incorrect expression pattern of the transgene in the non-GABAergic mitral and tufted cells of the OB of the transgenic line Tg(GAD67lacZ7.5)1. Here we use this transgenic line as a model to characterize the intracellular distribution of ß-galactosidase staining within the large output neurons at light microscopic and electron microscopic level by using ß-galactosidase enzyme and immunohistochemistry.
Light Microscopy
The laminar organization of the OB from the Tg(GAD67lacZ7.5)1 mouse line was indistinguishable from that of the OB from non-transgenic animals (Figure 1). The transgene expression was predominantly detected in the majority of mitral and tufted cells by the prominent bright blue X-gal reaction product, and occasionally in some granule and periglomerular cells (Figure 1 and Figure 2). The superficial tufted cells showed the strongest staining among the neuronal populations of the OB (Figure 2). Nuclei of mitral and tufted cells were strongly stained, whereas perikarya and dendrites of these cells were less intensely labeled. Diffuse staining without well-defined cell localization was seen in the glomeruli, the external plexiform layer (especially in its superficial segment), and the internal plexiform layer. In the granule cell layer, patches were labeled with diffuse blue staining. In addition, small punctate-like accumulation of the reaction product was observed in the perikarya and along the dendrites of the output neurons (Figure 2 and inset).
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Immunohistochemical detection of ß-galactosidase labeled the same subpopulation of neurons as revealed by enzyme histochemistry. The mitral and tufted cells were strongly stained and the OB was devoid of diffuse staining (Figure 3 Figure 4 Figure 5). The reaction product was mainly confined to the cytoplasm of neurons, although some nuclei were also stained (Figure 3 Figure 4 Figure 5). In addition to the perikarya, the dendrites and axons were also filled with reaction product (Figure 4). The strong dendritic staining was most evident in the superficial segment of the external plexiform layer, whereas the internal plexiform layer was slightly labeled (Figure 3 and Figure 4). The punctate-like accumulation of reaction product was also present in the cytoplasm of ß-galactosidase-positive neurons (Figure 4), as in Figure 2.
Electron Microscopy
Using electron microscopy, neurons expressing the LacZ fusion gene can be directly identified, because the 5-bromo-3-indolyl precipitate formed on cleavage of Bluo-gal by the ß-galactosidase reaction is highly electron-dense.
The product of the enzyme histochemical reaction was dispersed throughout the cytoplasm and nuclei of ß-galactosidase-positive neurons as submicroscopic grains with an average diameter of 30 nm. Within the same cells, large 0.2-0.5-µm-long electron-dense deposits of the reaction product were also present. Clusters of these electron-dense deposits were generally attached to intracellular membranes, including the nuclear envelope, mitochondria, and endoplasmic reticulum (Figure 6). On the somata of the labeled output neurons, several synapses were found with granule cells, although their presence was often masked by massive precipitate (Figure 6 and inset). Both types of precipitate were visible in the dendrites of the output neurons in the external plexiform layer (Figure 7), where dendro-dendritic synaptic contacts could be detected between labeled ß-galactosidase-positive output neurons and unlabeled ß-galactosidase-negative granule cells (Figure 7). The terminal segments of mitral/tufted cell dendrites contained very little reaction product (Figure 8). Because of the diffusion of the reaction product, we also found some precipitate in the extracellular space and in neighboring structures (Figure 7 and Figure 8).
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Immunohistochemical detection of ß-galactosidase resulted in a fine reaction product that almost completely filled the cytoplasm of neurons expressing the LacZ marker gene, including perikarya (Figure 9), dendrites (Figure 10), and axons (data not shown). The reaction product was also detected in some nuclei (Figure 9). In contrast to the enzyme histochemistry, cytoplasm of terminal dendrites of output neurons entering the olfactory glomeruli contained heavily electron-dense reaction product (Figure 11; compare to Figure 8). Several synaptic contacts were found between the labeled terminal segments of output neurons and axons of the unlabeled olfactory receptor neurons (Figure 11). Synaptic specializations were also identified on the somata as well as on the dendrites of labeled output neurons (Figure 9, Figure 12, and Figure 13). At high resolution, individual and reciprocal synaptic contacts between the labeled dendrites of mitral/tufted cells and the unlabeled ß-galactosidase-negative granule cells were readily detected (Figure 12 and Figure 13).
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Often, ß-galactosidase was found sequestered in large lysosome-like structures (Figure 6), which were identical to the punctate-like staining seen by light microscopy (Figure 2 and Figure 4).
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Discussion |
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In the OB of the transgenic mouse line Tg(GAD67-lacZ7.5)1, the majority of the mitral and tufted cells express the bacterial enzyme ß-galactosidase. This type of expression is regarded as incorrect (ectopic) because the large output neurons of the OB operate with glu-tamate rather than GABA as a neurotransmitter (
We have used ß-galactosidase enzyme and immunohistochemistry to identify ß-galactosidase-expressing output neurons and their synaptic connections in the OB at both light and electron microscopic levels. Using enzyme histochemistry, the blue reaction product accumulated mostly in neuronal nuclei, perikarya, and to some extent in the processes. Diffuse staining in the glomerular layer and external plexiform layer probably represents labeled processes of the output neurons. Especially strong staining of the superficial part of the external plexiform layer can be attributed to stained dendrites of the superficial tufted cells. On the other hand, the presence of diffuse patches in the internal plexiform and granule cell layers suggests axonal staining of the mitral and tufted cells. However, one cannot exclude the possibility that the diffuse staining is at least in part due to limitations of this method, i.e., diffusion of the reaction product out of the positive cells, as reported by
Diffusion was limited when Bluo-gal was used as a substrate, although X-gal was preferred for light microscopic visualization because it usually gave a brighter color reaction. Bluo-gal is a more suitable substrate for identifying ß-galactosidase-positive cells at the ultrastructural level than X-gal, as the 4-chloro-3-indolyl precipitate is more electron-dense and less diffusable. At the electron microscopic level, the Bluo-gal reaction product formed fine submicroscopic grains as well as larger electron-dense deposits bound to internal and occasionally to external membranes, which were in part also detected in some dendrites. The precipitate accumulating on the membranes makes it difficult to identify synaptic clefts on labeled neurons. In addition, diffusion of the reaction product into neighboring structures interferes with proper distinguishing between the terminal segments of the ß-galactosidase-positive output neurons with little reaction product and the ß-galactosidase-negative neuronal cells. Another obvious disadvantage of this detection system, reported also by
Comparison of staining of the mitral and tufted cells after enzyme histochemistry with that after immunohistochemistry revealed several advantages of using ß-galactosidase immunohistochemistry, at least for electron microscopic studies, most notably strict intracellular localization of the reaction product and homogeneous labeling of the entire cytoplasm, including processes and their terminal segments. Using immunohistochemistry for ß-galactosidase to label output neurons expressing LacZ enabled us to readily identify synaptic connections between the ß-galactosidase-negative olfactory receptor neurons and the labeled ß-galactosidase-positive mitral/tufted cells within the glomerular layer, as well as synaptic junctions between labeled soma/dendrite of mitral/tufted cells and ß-galactosidase-negative granule cells in the external plexiform layer.
The expression of ß-galactosidase in well-defined neuronal subpopulations in transgenic mouse lines carrying the LacZ reporter gene may prove useful in providing a neutral intrinsic marker for these neuronal populations that may help their identification after experimental manipulations such as transplantation, on ablation, and in cell culture. Here we show that this marker could successfully be used for identification of labeled neurons and their synaptic contacts with unlabeled neurons at the ultrastructural level, which may prove indispensible in cases where a specific marker is not available, as in the case of mitral and tufted neurons of the OB.
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Footnotes |
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1 Present address: Institute of Neuroscience, Northwestern University, Chicago, IL.
2 Deceased February 25, 1996.
3 On leave from the Institute of Biology, Romanian Academy of Sciences, Bucharest, Romania.
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Acknowledgments |
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Supported in part by the Hungarian Research Fund (OTKA T14645, T006373, and T016971) and the Ministry of Public Welfare (ETT T-04 029/93), by the Slovak Academy of Sciences grant No.1319, and by grants from the Deutsche Forschungsgemeinschaft SFB 406 and from the Volkswagen Stiftung (No. I-7-777).
We thank Ms Ildikó Harmos, Ms Annelies Wolff, and Martha Syneková for excellent technical assistance.
Received for publication October 15, 1996; accepted March 10, 1997.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arai Y, Kajihara S, Masuda J, Ohishi S, Zen K, Ogata J, Mukai T (1994) Position-independent, high-level, and correct regional expression of the rat aldolase C gene in the central nervous system of transgenic mice. Eur J Biochem 221:253-260 [Abstract]
Al-Shawi R, Kinnaird J, Burke J, Bishop JO (1990) Expression of a foreign gene in a line of transgenic mice is modulated by chromosal position effect. Mol Cell Biol 10:1192-1198 [Medline]
Banerjee SA, Hoppe P, Brilliant M, Chikaraishi DM (1992) 5' flanking sequences of the rat tyrosine hydroxylase gene target accurate tissue-specific, developmental, and transsynaptic expression in transgenic mice. J Neurosci 12:4460-4467 [Abstract]
Bessis A, Salmon A-M, Zoli M, Le Novere N, Picciotto M, Changeux J-P (1995) Promoter elements conferring neuron-specific expression of the ß2-subunit of the neuronal nicotinic acetylcholine receptor studied in vitro and in transgenic mice. Neuroscience 69:807-819 [Medline]
Charron G, Guy L-G, Bazinet M, Julien J-P (1995) Multiple neuron-specific enhancers in the gene coding for the human neurofilament light chain. J Biol Chem 270:30604-30610
í
ková D, Sekerková G, Oestreicher AB, Gispen WH,
igová T (1995) Distribution of growth associated protein (B-50/GAP-43) and glial fibrillary acidic protein (GFAP) immunoreactivity in rat homotopic olfactory bulb transplants. Arch Ital Biol 33:237-250
Cohen-Tannoudji M, Babinet C, Wassef M (1994) Early determination of a mouse somatosensory cortex marker. Nature 368:460-463 [Medline]
Erickson JC, Masters BA, Kelly EJ, Brinster RL, Palmiter RG (1995) Expression of human metallothionein-II in transgenic mice. Neurochem Int 27:35-41 [Medline]
Farbman AI (1992) Cell Biology of Olfaction. Cambridge, UK, Cambridge University Press
Forss-Petter S, Danielson PE, Catsicas S, Battenberg E, Price J, Nerenberg M, Sutcliffe JG (1990) Transgenic mice expressing ß-galactosidase in mature neurons under neuron-specific enolase promoter control. Neuron 5:187-197 [Medline]
Friedrich VL, Holstein GR, Li X, Gow A, Kelley KA, Lazzarini RA (1993) Intracellular distribution of transgenic bacterial beta- galactosidase in central nervous system neurons and neuroglia. J Neurosci Res 36:88-98 [Medline]
Graziadei PPC, Monti Graziadei AG (1979) Neurogenesis and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and structural organization of the olfactory sensory neurons. J Neurocytol 8:1-18 [Medline]
Halász N (1990) The Vertebrate Olfactory System. Budapest, Akadémiai Kiadó
Hoyle GW, Mercer EH, Palmiter RD, Brinster RL (1994) Cell-specific expression from the human dopamine ß-hydroxylase promoter in transgenic mice is controlled via a combination of positive and negative regulatory elements. J Neurosci 14:2455-2463 [Abstract]
Huh SO, Park DH, Cho JY, Joh TH, Son JH (1994) A 6.1kb 5' upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotoninergic brain regions and pineal gland in transgenic mice. Mol Brain Res 24:145-152 [Medline]
Lois C, Alvarez-Buylla A (1994) Long-distance neuronal migration in the adult mammalian brain. Science 264:1145-1148 [Medline]
Lois C, Garcia-Verdugo J-M, Alvarez-Buylla A (1996) Chain migration of neuronal precursors. Science 271:978-981 [Abstract]
Masters BA, Quaife CJ, Erickson JC, Kelly EJ, Froelick GJ, Zambrowicz BP, Brinster RL, Palmiter RD (1994) Metallothionein III is expressed in neurons that sequester zinc in synaptic vesicles. J Neurosci 14:5844-5857 [Abstract]
Min N, Joh TH, Kim KS, Peng C, Son JH (1994) 5' upstream DNA sequence of the rat tyrosine hydroxylase gene directs high-level and tissue specific expression to catecholaminergic neurons in the central nervous system of transgenic mice. Mol Brain Res 27:281-289 [Medline]
Monti Graziadei AG, Graziadei PPC (1984) The olfactory organ. In Sladek JR, Jr, Gash DM, eds. Neuronal Transplants. New York, Plenum Press, 167-185
Morgan WW, Walter CA, Windle JJ, Sharp ZD (1996) 3.6 kb of the 5' flanking DNA activates the mouse tyrosine hydroxylase gene promoter without catecholaminergic-specific expression. J Neurochem 66:20-25 [Medline]
Morita S, Kobayashi K, Mizuguchi T, Yamada K, Nagatsu I, Titani K, Fujita K, Hikada H, Nagatsu T (1993) The 5'-flanking region of the human dopamine ß-hydroxylase gene promotes neuron subtype-specific gene expression in the central nervous system of transgenic mice. Mol Brain Res 17:239-244 [Medline]
Mugnaini E, Oertel WH, Wouterlood FF (1984) Immunohistochemical localization of GABA neurons and dopamine neurons in the rat main and accessory olfactory bulb. Neurosci Lett 47:221-226 [Medline]
Ponte I, Martinez P, Ramirez A, Jorcano JL, Monzo M, Suau P (1994) Transcriptional activation of histone H1 zero during neuronal terminal differentiation. Dev Brain Res 80:35-44 [Medline]
Rincon-Limas DE, Geske RS, Xue J-J, Hsu CY, Overbeek PA, Patel PI (1994) 5'- flanking sequences of the human HPRT gene direct neuronal expression in the brain of transgenic mice. J Neurosci Res 38:259-267 [Medline]
Sanes JR, Rubenstein JLR, Nicolas J-F (1986) Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J 5:3133-3142 [Abstract]
Shepherd GM (1994) Discrimination of molecular signals by the olfactory receptor neuron. Neuron 13:771-790 [Medline]
Smeyne RJ, Curran T, Morgan JI (1992) Temporal and spatial expression of a fos-lacZ transgene in the developing nervous system. Mol Brain Res 16:158-162 [Medline]
Soriano E, Dumesnil N, Auladell C, Cohen-Tannoudji M, Sotelo C (1995) Molecular heterogeneity of progenitors and radial migration in the developing cerebral cortex revealed by transgene expression. Proc Natl Acad Sci USA 92:11676-11680 [Abstract]
Tan S-S, Breen S (1993) Radial mosaicism and tangential cell dispersion both contribute to mouse neocortical development. Nature 362:638-640 [Medline]
Vanselow J, Grabczyk E, Ping J, Beatscher M, Teng S, Fishman MC (1994) GAP-43 transgenic mice: dispersed genomic sequences confer a GAP-43-like expression pattern during development and regeneration. J Neurosci 14:499-510 [Abstract]
Weis J, Fine SM, David C, Savarirayan S, Sanes J (1991) Integration site-dependent expression of a transgene reveals specialized features of cells associated with neuromuscular junctions. J Cell Biol 113:1385-1397 [Abstract]
Wirak DO, Bayney R, Kundel CA, Lee A, Scangos GA, Trapp BD, Unterbeck AJ (1991) Regulatory region of human amyloid precursor protein (ASPP) gene promotes neuron-specific gene expression in the CNS of transgenic mice. EMBO J 10:289-296 [Abstract]