BAC transgenic mice express enhanced green fluorescent protein in central and peripheral cholinergic neurons

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 knock-in of eGFP within a bacterial artificial chromosome (BAC) spanning the ChAT locus and expression of this construct as a transgene. eGFP is expressed in ChAT(BAC)-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 postcoitus in the developing mouse embryo. ChAT(BAC)-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.

Selective degeneration of a physiological subtype of spinal motor neuron in mice with SOD1-linked ALS

“…To carry out whole cell patch-clamp recording, DIC imaging had to be guided by fluorescence microscopy. ChAT-EGFP mice, which express eGFP in cholinergic cells including MNs (17), or G85R SOD1YFP transgenic mice, which strongly express the YFP fusion protein in MNs (16), were thus used to produce slices with visible fluorescent MN pools in the expected anatomical locations in the ventral horn when observed with a 4× objective (e.g., Fig. 1A, eGFP, Left, and SI Discussion).…”

“…The mice paralyze uniformly by 5-6 mo of age. We first developed, using ChAT-EGFP mice that express GFP fluorescence in MNs (17), an acute slice preparation of adult mouse spinal cord that yielded healthy MNs in animals up to and beyond 6 mo of age, readily visualized by their fluorescence, enabling whole cell patch-clamp recordings when coupled with differential interference contrast (DIC) imaging. This preparation allowed extensive characterization of normal MN electrophysiology and…”

c-kit expression identifies cardiovascular precursors in the neonatal heart

“…C-kit expression appears to characterize a developmental stage of one subset of these cardiovascular precursor cells (1), but studies in mouse (4) and human (2, 3) ES cell-derived precursors suggest that a c-kit-negative stage precedes commitment. To examine the role of c-kit expression in CPcs, and to facilitate their isolation and analysis, we created BAC transgenic mice in which EGFP is placed under the transcriptional control of the c-kit locus to achieve high expression levels and outstanding transcriptional fidelity (20,21). Bernex et al (22) and Wouters et al (23) knocked reporters into the c-kit locus, but did not examine cardiac expression, and the dominant nature of the c-kit locus complicates this strategy.…”

Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes

“…Two independently generated ChAT (BAC) -eGFP mice were provided by M. Kotlikoff (Cornell University), and H. Monyer (University of Heidelberg) (10,11). Tg(Chrna3-EGFP)BZ135Gsat mice with eGFP driven by the nAChR α3β4α5 cluster were provided by I. Ibanez-Tallon (MDC Molecular Medicine) (24).…”