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The sustained expression of the MAFB transcription factor in human islet β-cells represents a distinct difference in mice. Moreover, mRNA expression of closely related and islet β-cell-enriched MAFA does not peak in humans until after 9 years of age. We show that the MAFA protein also is weakly produced within the juvenile human islet β-cell population and that expression is postnatally restricted in mouse β-cells by de novo DNA methylation. To gain insight into how MAFB affects human β-cells, we developed a mouse model to ectopically express in adult mouse β-cells using transcriptional control sequences. Coexpression of MafB with MafA had no overt impact on mouse β-cells, suggesting that the human adult β-cell MAFA/MAFB heterodimer is functionally equivalent to the mouse MafA homodimer. However, MafB alone was unable to rescue the islet β-cell defects in a mouse mutant lacking MafA in β-cells. Of note, transgenic production of MafB in β-cells elevated tryptophan hydroxylase 1 mRNA production during pregnancy, which drives the serotonin biosynthesis critical for adaptive maternal β-cell responses. Together, these studies provide novel insight into the role of MAFB in human islet β-cells.
© 2018 by the American Diabetes Association.
Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) mouse line with three catecholamine-related Cre recombinase mouse lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ∼ 6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines was generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing.
Published by Elsevier Ltd.
BACKGROUND - Unusually large CAG repeat expansions (>60) in exon one of Huntingtin (HTT) are invariably associated with a juvenile-onset form of Huntington's disease (HD), characterized by a more extensive and rapidly progressing neuropathology than the more prevalent adult-onset form. However, existing mouse models of HD that express the full-length Htt gene with CAG repeat lengths associated with juvenile HD (ranging between ~75 to ~150 repeats in published models) exhibit selective neurodegenerative phenotypes more consistent with adult-onset HD. Objective: To determine if a very large CAG repeat (>200) in full-length Htt elicits neurodegenerative phenotypes consistent with juvenile HD.
METHODS - Using a …bacterial artificial chromosome (BAC) system, we generated mice expressing full-length mouse Htt with ~225 CAG repeats under control of the mouse Htt promoter. Mice were characterized using behavioral, neuropathological, biochemical and brain imaging methods.
RESULTS - BAC-225Q mice exhibit phenotypes consistent with a subset of features seen in juvenile-onset HD: very early motor behavior abnormalities, reduced body weight, widespread and progressive increase in Htt aggregates, gliosis, and neurodegeneration. Early striatal pathology was observed, including reactive gliosis and loss of dopamine receptors, prior to detectable volume loss. HD-related blood markers of impaired energy metabolism and systemic inflammation were also increased. Aside from an age-dependent progression of diffuse nuclear aggregates at 6 months of age to abundant neuropil aggregates at 12 months of age, other pathological and motor phenotypes showed little to no progression.
CONCLUSIONS - The HD phenotypes present in animals 3 to 12 months of age make the BAC-225Q mice a unique and stable model of full-length mutant Htt associated phenotypes, including body weight loss, behavioral impairment and HD-like neurodegenerative phenotypes characteristic of juvenile-onset HD and/or late-stage adult-onset HD.
The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.
Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5'TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.
Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
The hemicholinium-3 (HC-3) sensitive, high-affinity choline transporter (CHT) sustains cholinergic signaling via the presynaptic uptake of choline derived from dietary sources or from acetylcholinesterase (AChE)-mediated hydrolysis of acetylcholine (ACh). Loss of cholinergic signaling capacity is associated with cognitive and motor deficits in humans and in animal models. Whereas genetic elimination of CHT has revealed the critical nature of CHT in maintaining ACh stores and sustaining cholinergic signaling, the consequences of elevating CHT expression have yet to be studied. Using bacterial artificial chromosome (BAC)-mediated transgenic methods, we generated mice with integrated additional copies of the mouse Slc5a7 gene. BAC-CHT mice are viable, appear to develop normally, and breed at wild-type (WT) rates. Biochemical studies revealed a 2 to 3-fold elevation in CHT protein levels in the CNS and periphery, paralleled by significant increases in [(3)H]HC-3 binding and synaptosomal choline transport activity. Elevations of ACh in the BAC-CHT mice occurred without compensatory changes in the activity of either choline acetyltransferase (ChAT) or AChE. Immunohistochemistry for CHT in BAC-CHT brain sections revealed markedly elevated CHT expression in the cell bodies of cholinergic neurons and in axons projecting to regions known to receive cholinergic innervation. Behaviorally, BAC-CHT mice exhibited diminished fatigue and increased speeds on the treadmill test without evidence of increased strength. Finally, BAC-CHT mice displayed elevated horizontal activity in the open field test, diminished spontaneous alteration in the Y-maze, and reduced time in the open arms of the elevated plus maze. Together, these studies provide biochemical, pharmacological and behavioral evidence that CHT protein expression and activity can be elevated beyond that seen in wild-type animals. BAC-CHT mice thus represent a novel tool to examine both the positive and negative impact of constitutively elevated cholinergic signaling capacity.
Copyright © 2013 Elsevier Ltd. All rights reserved.
Uchl1 encodes the protein gene product 9.5 antigen (PGP9.5) that is a widely used to identify migrating neural progenitors in the PNS, mature neurons of the central and peripheral nervous systems, as well as neuroendocrine cells. To facilitate analysis of developing peripheral neurons, we linked regulatory regions of Uchl1 carried within a 160kb bacterial artificial chromosome (BAC) to the dual fluorescent reporter H2BmCherry:GFP-gpi. The Uchl1-H2BmCherry:GFP-gpi transgene exhibits robust expression and allows clear discrimination of individual cells and cellular processes in cranial ganglia, sympathetic chain, the enteric nervous system (ENS), and autonomic ganglia of the urogenital system. The transgene also labels subsets of cells in endocrine tissues where earlier in situ hybridization (ISH) studies have previously identified expression of this deubiquinating enzyme. The Uchl1-H2BmCherry:GFP-gpi transgene will be a powerful tool for static and live imaging, as well as isolation of viable neural progenitors to investigate processes of autonomic neurogenesis.
Copyright © 2013 Wiley Periodicals, Inc.
We describe the first example of combining bacterial artificial chromosome (BAC) recombination-mediated mutagenesis with reverse genetics for a negative strand RNA virus. A BAC-based respiratory syncytial virus (RSV) rescue system was established. An important advantage of this system is that RSV antigenomic cDNA was stabilized in the BAC vector. The RSV genotype chosen was A2-line19F, a chimeric strain previously shown to recapitulate in mice key features of RSV pathogenesis. We recovered two RSV reporter viruses, one expressing the red fluorescent protein monomeric Katushka 2 (A2-K-line19F) and one expressing Renilla luciferase (A2-RL-line19F). As proof of principle, we efficiently generated a RSV gene deletion mutant (A2-line19FΔNS1/NS2) and a point mutant (A2-K-line19F-I557V) by recombination-mediated BAC mutagenesis. Together with sequence-optimized helper expression plasmids, BAC-RSV is a stable, versatile, and efficient reverse genetics platform for generation of a recombinant Pneumovirus.
Copyright © 2012 Elsevier Inc. All rights reserved.
Certain groups of physically linked genes remain linked over long periods of evolutionary time. The general view is that such evolutionary conservation confers 'fitness' to the species. Why gene order confers 'fitness' to the species is incompletely understood. For example, linkage of IL26 and IFNG is preserved over evolutionary time yet Th17 lineages express IL26 and Th1 lineages express IFNG. We considered the hypothesis that distal enhancer elements may be shared between adjacent genes, which would require linkage be maintained in evolution. We test this hypothesis using a bacterial artificial chromosome transgenic model with deletions of specific conserved non-coding sequences. We identify one enhancer element uniquely required for IL26 expression but not for IFNG expression. We identify a second enhancer element positioned between IL26 and IFNG required for both IL26 and IFNG expression. One function of this enhancer is to facilitate recruitment of RNA polymerase II to promoters of both genes. Thus, sharing of distal enhancers between adjacent genes may contribute to evolutionary preservation of gene order.
The intronic GABRG2 mutation, IVS6+2T→G, was identified in an Australian family with childhood absence epilepsy and febrile seizures (Kananura et al., 2002). The GABRG2 intron 6 splice donor site was found to be mutated from GT to GG. We generated wild-type and mutant γ2 subunit bacterial artificial chromosomes (BACs) driven by a CMV promoter and expressed them in HEK293T cells and expressed wild-type and mutant γ2 subunit BACs containing the endogenous hGABRG2 promoter in transgenic mice. Wild-type and mutant GABRG2 mRNA splicing patterns were determined in both BAC-transfected HEK293T cells and transgenic mouse brain, and in both, the mutation abolished intron 6 splicing at the donor site, activated a cryptic splice site, generated partial intron 6 retention, and produced a frameshift in exon 7 that created a premature translation termination codon (PTC). The resultant mutant mRNA was either degraded partially by nonsense-mediated mRNA decay or translated to a stable, truncated subunit (the γ2-PTC subunit) containing the first six GABRG2 exons and a novel frameshifted 29 aa C-terminal tail. The γ2-PTC subunit was homologous to the mollusk AChBP (acetylcholine binding protein) but was not secreted from cells. It was retained in the ER and not expressed on the surface membrane, but it did oligomerize with α1 and β2 subunits. These results suggested that the GABRG2 mutation, IVS6+2T→G, reduced surface αβγ2 receptor levels, thus reducing GABAergic inhibition, by reducing GABRG2 transcript level and producing a stable, nonfunctional truncated subunit that had a dominant-negative effect on αβγ2 receptor assembly.