Mouse Bacterial Artificial Chromosome Research Articles

Effect of aging and exercise on hTERT expression in thymus tissue of hTERT transgenic bacterial artificial chromosome mice.

Telomere shortening occurs with aging in immune cells and may be related to immunosenescence. Exercise can upregulate telomerase activity and attenuate telomere shortening in immune cells, but it is unknown if exercise impacts other immune tissues such as the thymus. This study aimed to examine human telomerase reverse transcriptase (hTERT) alternative splicing (AS) in response to aging and exercise in thymus tissue. Transgenic mice with a human TERT bacterial artificial chromosome integrated into its genome (hTERT-BAC) were utilized in two different exercise models. Mice of different ages were assigned to an exercise cage (running wheel) or not for 3weeks prior to thymus tissue excision. Middle-aged mice (16months) were exposed or not to treadmill running (30min at 60% maximum speed) prior to thymus collection. hTERT transcript variants were measured by RT-PCR. hTERT transcripts decreased with aging (r = - 0.7511, p < 0.0001) and 3weeks of wheel running did not counteract this reduction. The ratio of exons 7/8 containing hTERT to total hTERT transcripts increased with aging (r = 0.3669, p = 0.0423) but 3weeks of voluntary wheel running attenuated this aging-driven effect (r = 0.2013, p = 0.4719). Aging increased the expression of senescence marker p16 with no impact of wheel running. Thymus regeneration transcription factor, Foxn1, went down with age with no impact of wheel running exercise. Acute treadmill exercise did not induce any significant changes in thymus hTERT expression or AS variant ratio (p > 0.05). In summary, thymic hTERT expression is reduced with aging. Exercise counteracted a shift in hTERT AS ratio with age. Our data demonstrate that aging impacts telomerase expression and that exercise impacts dysregulated splicing that occurs with aging.

Maintenance of optogenetic channel rhodopsin (ChR2) function in aging mice: Implications for pharmacological studies of inhibitory synaptic transmission, quantal content, and calcium homeostasis

Disruption of synaptic function is believed to represent a common pathway contributing to cognitive decline during aging. Optogenetics is a prodigious tool for studying relationships between function and synaptic circuitry but models utilizing viral vectors present limitations. Careful characterization of the functionality of channel rhodopsin in transgenic models is crucial for determining whether they can be used across aging. This includes verifying the light sensitivity of the protein and confirming its ability to generate action potentials in response to light stimulation. We combined in vitro optogenetic methodology and a reduced synaptic preparation of acutely isolated neurons to determine if the ChR2(H134R)-eYFP vGAT mouse model is well-suited for aging studies. We used neurons from young (2–6 mo), middle-aged (10–14 mo) and aged (17–25 mo) bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R in GABAergic cell populations. Cellular physiology and calcium dynamics were assessed in basal forebrain (BF) neurons using patch-clamp recording and fura-2 microfluorimetry, alongside 470 nm light stimulation of the transgenic ChR2 channel to characterize a wide array of physiological functions known to decline with age. We found ChR2 expression is functionally maintained across aging, while spontaneous and optically evoked inhibitory postsynaptic currents, and quantal content were decreased. Aged mice also showed an increase in intracellular calcium buffering. These results, which are on par with previous observations, demonstrate that the optogenetic vGAT BAC mouse model is well-suited for investigating age-related changes in calcium signaling and synaptic transmission.

Acute Exercise Regulates hTERT Gene Expression and Alternative Splicing in the hTERT-BAC Transgenic Mouse Model.

Aerobic exercise maintains telomere length through increased human telomerase reverse transcriptase (hTERT) expression and telomerase enzyme activity. The impact of acute exercise on hTERT alternative splicing (AS) is unknown. This study aimed to examine hTERT AS in response to acute treadmill running. A bacterial artificial chromosome mouse model containing the 54-kilobase hTERT gene locus inserted into its genome (hTERT-BAC) was utilized. The gastrocnemius, left ventricle, and brain were excised before (Pre), upon cessation (Post), and during recovery (1, 24, 48, and 72 h; n = 5/time point) from treadmill running (30 min at 60% maximum speed). Full-length (FL) hTERT and the "minus beta" (-β) AS variant (skips exons 7 and 8 and does not code for active telomerase) were measured by gel-based and droplet digital reverse transcription-polymerase chain reaction methods. SF3B4 and SRSF2 protein expression were measured by Western blotting. Compared with Pre, FL hTERT increased at Post before decreasing during recovery in the gastrocnemius (48 and 72 h; P ≤ 0.001) and left ventricle (24 h; P = 0.004). The percentage of FL hTERT in the gastrocnemius also increased during recovery (1 and 72 h; P ≤ 0.017), whereas a decrease was observed in the left ventricle (1, 24, and 48 h; P ≤ 0.041). hTERT decreased in the brain (48 h), whereas FL hTERT percentage remained unaltered. SF3B4 protein expression decreased throughout recovery in the gastrocnemius and tended to be associated with FL hTERT (r = -0.348, P = 0.075) and -β in opposite directions (r = 0.345, P = 0.067). Endurance exercise increased hTERT gene expression, and altered FL hTERT splicing in contractile tissues and may maintain telomere length necessary to improve the function and health of the organism.

Uninterrupted CAG repeat drives striatum-selective transcriptionopathy and nuclear pathogenesis in human Huntingtin BAC mice

In Huntington's disease (HD), the uninterrupted CAG repeat length, but not the polyglutamine length, predicts disease onset. However, the underlying pathobiology remains unclear. Here, we developed bacterial artificial chromosome (BAC) transgenic mice expressing human mutant huntingtin (mHTT) with uninterrupted, and somatically unstable, CAG repeats that exhibit progressive disease-related phenotypes. Unlike prior mHTT transgenic models with stable, CAA-interrupted, polyglutamine-encoding repeats, BAC-CAG mice show robust striatum-selective nuclear inclusions and transcriptional dysregulation resembling those in murine huntingtin knockin models and HD patients. Importantly, the striatal transcriptionopathy in HD models is significantly correlated with their uninterrupted CAG repeat length but not polyglutamine length. Finally, among the pathogenic entities originating from mHTT genomic transgenes and only present or enriched in the uninterrupted CAG repeat model, somatic CAG repeat instability and nuclear mHTT aggregation are best correlated with early-onset striatum-selective molecular pathogenesis and locomotor and sleep deficits, while repeat RNA-associated pathologies and repeat-associated non-AUG (RAN) translation may play less selective or late pathogenic roles, respectively.

Acute Exercise Regulates Htert Alternative Splicing In Contractile Tissues Of Htert-bac Mice

Full length human telomerase reverse transcriptase (FL hTERT) is the rate-limiting, catalytic component necessary for telomerase enzyme activity and telomere length maintenance. Unlike rodents, humans evolved elongated introns with cis-elements that when bound by specific RNA binding proteins (RNAbp) splice hTERT to FL transcripts or produce inactive variants through alternative splicing (AS). Our working hypothesis is that hTERT AS regulates the amount of active telomerase. Evidence from human studies have shown that aerobic exercise maintains telomere length through increased hTERT expression and telomerase enzyme activity, however the impact of acute exercise on hTERT AS is unknown. PURPOSE: To examine hTERT AS regulation in response to acute treadmill running. METHODS: A bacterial artificial chromosome mouse model containing the full hTERT gene (5' and 3' regulatory elements; 16 exons and 15 introns) inserted into its genome (hTERT-BAC) was utilized. Gastrocnemius (Gas.) and heart (Hrt.) were excised from 10-week old hTERT-BAC mice prior to (PRE), upon cessation (POST), and during recovery (1, 24, 48, and 72H; n = 5/time point) from 30 min. of treadmill running (60% max. speed). Primers specific to hTERT were utilized to measure FL and the most common AS variant minus beta (-β, which skips exons 7 and 8) by two different RT-PCR methods - gel based and droplet digital PCR. Gene expression of the RNAbp PTBP1 was also measured. RESULTS: The expected human patterns of hTERT AS was recapitulated across all tissues examined. Compared to PRE, FL hTERT gene expression increased at POST before decreasing in the Gas. (48 and 72H; p ≤ 0.001) and Hrt. (24H; p = 0.004). Acute exercise also increased the percentage of FL hTERT in the Gas. at 1 and 72H (p ≤ 0.017), whereas a decrease was observed in the Hrt. at 1, 24, and 48H (p ≤ 0.041). Finally, Ptbp1 decreased at POST (Gas.) and at 1, 24, 48, and 72H (Gas. and Hrt.; p ≤ 0.05). CONCLUSIONS: Evolutionarily conserved mouse splicing machinery (RNAbp) recognize hTERT cis-elements to recapitulate human AS patterns and highlight the importance of the intronic elements in regulating hTERT splicing choice in humans. The hTERT-BAC mouse is a valuable resource to study the exercise-induced adaptive mechanisms that maintain telomere lengths in endurance trained individuals across the lifespan.

ALS-associated genes in SCA2 mouse spinal cord transcriptomes.

The spinocerebellar ataxia type 2 (SCA2) gene ATXN2 has a prominent role in the pathogenesis and treatment of amyotrophic lateral sclerosis (ALS). In addition to cerebellar ataxia, motor neuron disease is often seen in SCA2, and ATXN2 CAG repeat expansions in the long normal range increase ALS risk. Also, lowering ATXN2 expression in TDP-43 ALS mice prolongs their survival. Here we investigated the ATXN2 relationship with motor neuron dysfunction in vivo by comparing spinal cord (SC) transcriptomes reported from TDP-43 and SOD1 ALS mice and ALS patients with those from SCA2 mice. SC transcriptomes were determined using an SCA2 bacterial artificial chromosome mouse model expressing polyglutamine expanded ATXN2. SCA2 cerebellar transcriptomes were also determined, and we also investigated the modification of gene expression following treatment of SCA2 mice with an antisense oligonucleotide (ASO) lowering ATXN2 expression. Differentially expressed genes (DEGs) defined three interconnected pathways (innate immunity, fatty acid biosynthesis and cholesterol biosynthesis) in separate modules identified by weighted gene co-expression network analysis. Other key pathways included the complement system and lysosome/phagosome pathways. Of all DEGs in SC, 12.6% were also dysregulated in the cerebellum. Treatment of mice with an ATXN2 ASO also modified innate immunity, the complement system and lysosome/phagosome pathways. This study provides new insights into the underlying molecular basis of SCA2 SC phenotypes and demonstrates annotated pathways shared with TDP-43 and SOD1 ALS mice and ALS patients. It also emphasizes the importance of ATXN2 in motor neuron degeneration and confirms ATXN2 as a therapeutic target.

Human IL10 promoter haplotypes confer differential outcomes to experimental colitis in humanized BAC mice

Abstract Most genetic variants associated with complex diseases map to non-coding regions of the genome, but determining their phenotypic consequences remains challenging. We devised a bacterial artificial chromosome (BAC) transgenic approach to model the effects of human non-coding single nucleotide polymorphisms (ncSNPs) on complex disease outcomes. As proof-of-concept, we focus on human IL10, a potent anti-inflammatory cytokine and key mediator of gut homeostasis. ncSNPs in the IL10 locus have been strongly associated with a variety of disease risks, including colitis susceptibility, but their biological functions are unclear. We created two genetically humanized IL10BAC transgenic mouse lines based on two common and well-defined −1082/−819/−592 IL10 promoter haplotypes (“ATA” and “GCC”) on a mouse Il10-deficient background. Although both IL10BAC strains were protected from spontaneous colitis, only ATA-bearing mice were resistant to dextran sulfate sodium (DSS)-induced acute colitis, while GCC and WT mice had high rates of morbidity and mortality. We found that ATAs had different kinetics of cell-type-specific IL-10 production pre- and post-DSS treatment compared to GCCs and WTs, which influenced the expression profiles of other cytokines and led to distinct immunophenotypes. The skewed responses culminated in the inability of GCCs and WTs to control DSS-induced bacteremia, resulting in death. Our data suggest that human IL10 ncSNPs impart allele-specific differences in spatiotemporal regulation of IL-10 expression, leading to differential outcomes to experimental colitis. Humanized BAC mice may serve as an important tool in modeling the potential impact of human non-coding variants on complex biological outcomes.

Impact of broad regulatory regions on Gdf5 expression and function in knee development and susceptibility to osteoarthritis

ObjectivesGiven the role of growth and differentiation factor 5 (GDF5) in knee development and osteoarthritis risk, we sought to characterise knee defects resulting from Gdf5 loss of function and how...

A novel dopamine transporter transgenic mouse line for identification and purification of midbrain dopaminergic neurons reveals midbrain heterogeneity.

Midbrain dopaminergic (DAergic) neurons are a heterogeneous cell group, composed of functionally distinct cell populations projecting to the basal ganglia, prefrontal cortex and limbic system. Despite their functional significance, the midbrain population of DAergic neurons is sparse, constituting only 20000-30000 neurons in mice, and development of novel tools to identify these cells is warranted. Here, a bacterial artificial chromosome mouse line [Dat1-enhanced green fluorescent protein (eGFP)] from the Gene Expression Nervous System Atlas (GENSAT) that expresses eGFP under control of the dopamine transporter (DAT) promoter was characterized. Confocal microscopy analysis of brain sections showed strong eGFP signal reporter in midbrain regions and striatal terminals that co-localized with the DAergic markers DAT and tyrosine hydroxylase (TH). Thorough quantification of co-localization of the eGFP reporter signal with DAT and TH in the ventral midbrain showed that a vast majority of eGFP-expressing neurons are DAergic. Importantly, expression profiles also revealed DAergic heterogeneity when comparing substantia nigra and ventral tegmental area. Dat1-eGFP mice showed neither change in synaptosomal DA uptake nor altered levels of DAT and TH in both striatum and midbrain. No behavioural difference between Dat1-eGFP and wild-type was found, suggesting that the strain is not aberrant. Finally, cell populations highly enriched in DAergic neurons can be obtained from postnatal mice by fluorescence-activated cell sorting and the sorted neurons can be cultured invitro. The current investigation demonstrates that eGFP expression in this mouse line is selective for DAergic neurons, suggesting that the Dat1-eGFP mouse strain constitutes a promising tool for delineating new aspects of DA biology.

NIG_MoG: a mouse genome navigator for exploring intersubspecific genetic polymorphisms.

The National Institute of Genetics Mouse Genome database (NIG_MoG; http://molossinus.lab.nig.ac.jp/msmdb/) primarily comprises the whole-genome sequence data of two inbred mouse strains, MSM/Ms and JF1/Ms. These strains were established at NIG and originated from the Japanese subspecies Mus musculus molossinus. NIG_MoG provides visualized genome polymorphism information, browsing single-nucleotide polymorphisms and short insertions and deletions in the genomes of MSM/Ms and JF1/Ms with respect to C57BL/6J (whose genome is predominantly derived from the West European subspecies M. m. domesticus). This allows users, especially wet-lab biologists, to intuitively recognize intersubspecific genome divergence in these mouse strains using visual data. The database also supports the in silico screening of bacterial artificial chromosome (BAC) clones that contain genomic DNA from MSM/Ms and the standard classical laboratory strain C57BL/6N. NIG_MoG is thus a valuable navigator for exploring mouse genome polymorphisms and BAC clones that are useful for studies of gene function and regulation based on intersubspecific genome divergence.

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a ‘targeting’ vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Electrophysiological properties of genetically identified subtypes of layer 5 neocortical pyramidal neurons: Ca²⁺ dependence and differential modulation by norepinephrine.

We studied neocortical pyramidal neurons from two lines of bacterial artificial chromosome mice (etv1 and glt; Gene Expression Nervous System Atlas: GENSAT project), each of which expresses enhanced green fluorescent protein (EGFP) in a different subpopulation of layer 5 pyramidal neurons. In barrel cortex, etv1 and glt pyramidal cells were previously reported to differ in terms of their laminar distribution, morphology, thalamic inputs, cellular targets, and receptive field size. In this study, we measured the laminar distribution of etv1 and glt cells. On average, glt cells were located more deeply; however, the distributions of etv1 and glt cells extensively overlap in layer 5. To test whether these two cell types differed in electrophysiological properties that influence firing behavior, we prepared acute brain slices from 2-4-wk-old mice, where EGFP-positive cells in somatosensory cortex were identified under epifluorescence and then studied using whole cell current- or voltage-clamp recordings. We studied the details of action potential parameters and repetitive firing, characterized by the larger slow afterhyperpolarizations (AHPs) in etv1 neurons and larger medium AHPs (mAHPS) in glt cells, and compared currents underlying the mAHP and slow AHP (sAHP) in etv1 and glt neurons. Etv1 cells exhibited lower dV/dt for spike polarization and repolarization and reduced direct current (DC) gain (lower f-I slope) for repetitive firing than glt cells. Most importantly, we found that 1) differences in the expression of Ca(2+)-dependent K(+) conductances (small-conductance calcium-activated potassium channels and sAHP channels) determine major functional differences between etv1 and glt cells, and 2) there is differential modulation of etv1 and glt neurons by norepinephrine.

Novel BAC Mouse Model of Huntington's Disease with 225 CAG Repeats Exhibits an Early Widespread and Stable Degenerative Phenotype

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. 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. 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. 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.

Differential LRRK2 expression in the cortex, striatum, and substantia nigra in transgenic and nontransgenic rodents

ABSTRACTMutations in leucine‐rich repeat kinase 2 (LRRK2) are found in a significant proportion of late‐onset Parkinson's disease (PD) patients. Elucidating the neuroanatomical localization of LRRK2 will further define LRRK2 function and the molecular basis of PD. Here, we utilize recently characterized monoclonal antibodies to evaluate LRRK2 expression in rodent brain regions relevant to PD. In both mice and rats, LRRK2 is highly expressed in the cortex and striatum, particularly in pyramidal neurons of layer V and in medium spiny neurons within striosomes. Overall, rats have a more restricted distribution of LRRK2 compared with mice. Mice, but not rats, show high levels of LRRK2 expression in the substantia nigra pars compacta. Expression of the pathogenic LRRK2‐G2019S protein from mouse bacterial artificial chromosome (BAC) constructs closely mimics endogenous LRRK2 distribution in the mouse brain. However, LRRK2‐G2019S expression derived from human BAC constructs causes LRRK2 to be expressed in additional neuron subtypes in the rat such as striatal cholinergic interneurons and the substantia nigra pars compacta. The distribution of LRRK2 from human BAC constructs more closely resembles descriptions of LRRK2 in humans and nonhuman primates. Computational analyses of DNA regulatory elements in LRRK2 show a primate‐specific promoter sequence that does not exist in lower mammalian species. These noncoding regions may be involved in directing neuronal expression patterns. Together, these studies will aid in understanding the normal function of LRRK2 in the brain and will assist in model selection for future studies. J. Comp. Neurol. 522:2465–2480, 2014. © 2014 Wiley Periodicals, Inc.

Differential LRRK2 expression in the cortex, striatum, and substantia nigra in transgenic and nontransgenic rodents

Mutations in leucine-rich repeat kinase 2 (LRRK2) are found in a significant proportion of late-onset Parkinson's disease (PD) patients. Elucidating the neuroanatomical localization of LRRK2 will further define LRRK2 function and the molecular basis of PD. Here, we utilize recently characterized monoclonal antibodies to evaluate LRRK2 expression in rodent brain regions relevant to PD. In both mice and rats, LRRK2 is highly expressed in the cortex and striatum, particularly in pyramidal neurons of layer V and in medium spiny neurons within striosomes. Overall, rats have a more restricted distribution of LRRK2 compared with mice. Mice, but not rats, show high levels of LRRK2 expression in the substantia nigra pars compacta. Expression of the pathogenic LRRK2-G2019S protein from mouse bacterial artificial chromosome (BAC) constructs closely mimics endogenous LRRK2 distribution in the mouse brain. However, LRRK2-G2019S expression derived from human BAC constructs causes LRRK2 to be expressed in additional neuron subtypes in the rat such as striatal cholinergic interneurons and the substantia nigra pars compacta. The distribution of LRRK2 from human BAC constructs more closely resembles descriptions of LRRK2 in humans and nonhuman primates. Computational analyses of DNA regulatory elements in LRRK2 show a primate-specific promoter sequence that does not exist in lower mammalian species. These noncoding regions may be involved in directing neuronal expression patterns. Together, these studies will aid in understanding the normal function of LRRK2 in the brain and will assist in model selection for future studies.

A novel mouse model for Down syndrome that harbor a single copy of human artificial chromosome (HAC) carrying a limited number of genes from human chromosome 21

Down syndrome (DS), also known as Trisomy 21, is the most common chromosome aneuploidy in live-born children and displays a complicated symptom. To date, several kinds of mouse models have been generated to understand the molecular pathology of DS, yet the gene dosage effects and gene(s)-phenotype(s) correlation are not well understood. In this study, we established a novel method to generate a partial trisomy mice using the mouse ES cells that harbor a single copy of human artificial chromosome (HAC), into which a small human DNA segment containing human chromosome 21 genes cloned in a bacterial artificial chromosome (BAC) was recombined. The produced mice were found to maintain the HAC carrying human genes as a mini-chromosome, hence termed as a Trans-Mini-Chromosomal (TMC) mouse, and HAC was transmitted for more than twenty generations independent from endogenous mouse chromosomes. The three human transgenes including cystathionine β-synthase, U2 auxiliary factor and crystalline alpha A were expressed in several mouse tissues with various expression levels relative to mouse endogenous genes. The novel system is applicable to any of human and/or mouse BAC clones. Thus, the TMC mouse carrying a HAC with a limited number of genes would provide a novel tool for studying gene dosage effects involved in the DS molecular pathogenesis and the gene(s)-phenotype(s) correlation.

A Novel BACHD Transgenic Rat Exhibits Characteristic Neuropathological Features of Huntington Disease

Huntington disease (HD) is an inherited progressive neurodegenerative disorder, characterized by motor, cognitive, and psychiatric deficits as well as neurodegeneration and brain atrophy beginning in the striatum and the cortex and extending to other subcortical brain regions. The genetic cause is an expansion of the CAG repeat stretch in the HTT gene encoding huntingtin protein (htt). Here, we generated an HD transgenic rat model using a human bacterial artificial chromosome (BAC), which contains the full-length HTT genomic sequence with 97 CAG/CAA repeats and all regulatory elements. BACHD transgenic rats display a robust, early onset and progressive HD-like phenotype including motor deficits and anxiety-related symptoms. In contrast to BAC and yeast artificial chromosome HD mouse models that express full-length mutant huntingtin, BACHD rats do not exhibit an increased body weight. Neuropathologically, the distribution of neuropil aggregates and nuclear accumulation of N-terminal mutant huntingtin in BACHD rats is similar to the observations in human HD brains. Aggregates occur more frequently in the cortex than in the striatum and neuropil aggregates appear earlier than mutant htt accumulation in the nucleus. Furthermore, we found an imbalance in the striatal striosome and matrix compartments in early stages of the disease. In addition, reduced dopamine receptor binding was detectable by in vivo imaging. Our data demonstrate that this transgenic BACHD rat line may be a valuable model for further understanding the disease mechanisms and for preclinical pharmacological studies.

Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2

Kinesin-5 is an essential mitotic motor. However, how its spatial-temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification-tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end-directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end-directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2.

Generation of a model of l-DOPA-induced dyskinesia in two different mouse strains

Prolonged use of the dopamine precursor l-DOPA for the treatment of Parkinson's disease commonly results in abnormal involuntary movements, which are termed l-DOPA-induced dyskinesia (LID). Over-activity at corticostriatal synapses onto neurons of the direct and indirect striatal output pathways has been implicated in the development of dyskinesia, but it has proved difficult to investigate the pathways separately due to their morphological similarities. The recent development of bacterial artificial chromosome mice that express green fluorescent protein in either the direct or indirect pathway allows visual identification of the output neurons in each pathway.Here we describe the use of two different strains of these transgenic mice (pure FVB and FVB crossed with C57B1/6) in the development of mouse models of l-DOPA-induced dyskinesia. This model will allow the direct and indirect pathways to be studied individually to delineate the cellular and molecular mechanisms underlying dyskinesias. These studies demonstrate that mouse strain impacts on behavioural responses and l-DOPA sensitivity. Therefore, when generating mouse models of LID, strain must be taken into consideration when choosing the l-DOPA dosing regimen.

A new role for the extracellular calcium-sensing receptor demonstrated by using CCK-eGFP BAC mice

cesses both exciting and fun is the application of new techniques to address a problem where understanding has been constrained by previous techniques. Two complementary papers (10, 19) in this issue illustrate such an application. The new technique is the use of bacterial artificial chromosome (BAC) transgenic mice. The problem is nutrient sensing by enteroendocrine cells. Widely used in neuroscience research (5), the BAC mice allow either targeted gene deletion or overexpression (20). Many transgenic BAC mice express regulatory sequences together with enhanced green fluorescent protein (eGFP), which also allows identification of specific cell types. The eGFP allows these cells to be recovered using FACS for further analysis. Such BAC transgenic mice may then be mated with knockout or reporter mice to further define the presence of either a receptor or a pathway activated by the receptor. The satiating effects of dietary protein may be due in part to cholecystokinin (CCK) secretion from duodenal I cells. Although aromatic amino acids have been understood to be mediators of CCK secretion from these cells, the technical difficulties of recovering sufficient amounts of homogeneous I cells from fetal or adult intestine are character building. The use of cell lines (i.e., STC-1) that are tumorigenic but endocrine derived is informative but equivocal. Consequently, little is understood about the cellular mechanism by which amino acids affect CCK secretion. The extracellular calcium-sensing receptor (CaSR), originally cloned from the parathyroid, but found in distinct regions of the gastrointestinal system, may be activated by aromatic amino acids, consistent with the CaSR being an amino acid sensor (1, 2). Because the CaSR knockout is perinatal lethal, it was “rescued” by taking parathyroid hormone (PTH) out of the system through breeding with CaSR / PTH / heterozygotes. The resulting CaSR / PTH / are sustained with 2% Ca 2 in their drinking water (8, 9). Analyses using heterologous CaSR expressing cells have shown differences in intracellular Ca 2 oscillation frequency (13, 14) generated by aromatic amino acids compared with other ligands, but direct in vivo evidence from enteroendocrine cells has been lacking. Indeed, we still don’t understand the physiological importance of circulating amino acids in regulating the CaSR’s activity outside the gastrointestinal tract (3). The study by Liou et al. (10) from the laboratories of Raybould and Wank is as impressive as it is elegant. They used FACS to isolate and enrich duodenal I cells from the CCKeGFP BAC transgenic mice and demonstrated that the CaSR