Bacterial Artificial Chromosome Transgene Research Articles

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation

Single domain antibodies (sdAbs) correspond to the antigen-binding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ∼300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ∼1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.

GATA-related hematologic disorders.

The transcription factors GATA1 and GATA2 are fundamental regulators of hematopoiesis and have overlapping expression profiles. GATA2 is expressed in hematopoietic stem cells and early erythroid-megakaryocytic progenitors and activates a certain set of early-phase genes, including the GATA2 gene itself. GATA2 also initiates GATA1 gene expression. In contrast, GATA1 is expressed in relatively mature erythroid progenitors and facilitates the expression of genes associated with differentiation, including the GATA1 gene itself; however, GATA1 represses the expression of GATA2. Switching the GATA factors from GATA2 to GATA1 appears to be one of the key regulatory mechanisms underlying erythroid differentiation. Loss-of-function analyses using mice invivo have indicated that GATA2 and GATA1 are functionally nonredundant and that neither can compensate for the absence of the other. However, transgenic expression of GATA2 under the transcriptional regulation of the Gata1 gene rescues lethal dyserythropoiesis in GATA1-deficient mice, illustrating that the dynamic expression profiles of these GATA factors are critically important for the maintenance of hematopoietic homeostasis. Analysis of naturally occurring leukemias in GATA1-knockdown mice revealed that leukemic stem cells undergo functional alterations in response to exposure to chemotherapeutic agents. This mechanism may also underlie the aggravating features of relapsing leukemias. Recent hematologic analyses have suggested that disturbances in the balance of the GATA factors are associated with specific types of hematopoietic disorders. Here, we describe GATA1- and GATA2-related hematologic diseases, focusing on the regulation of GATA factor gene expression.

Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice.

Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a “C” or a “T” at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency phenotypes, but did not reveal an impact of the rs11115 variant on DBH expression in mice.

The untapped potential of the GENSAT mice-A valuable resource for developmental biology.

Gene Expression Nervous System Atlas (GENSAT) transgenic mice express EGFP, tdTomato, or Cre recombinase in a wide range of cell types. The mice and the bacterial artificial chromosome transgenes are available from repositories (MMRRC or CHORI), thereby making these resources readily available to the research community. This resource of 1,386 transgenic lines was developed and validated for neuroscience research. However, GENSAT mice have many potential applications in other contexts including studies of development outside of the CNS. The cell type-specific expression of fluorescent proteins in these mice has been used to identify cells in living embryos, in living embryo explants, and in stem or progenitor cell populations in postnatal tissues. The large number of fluorescent protein driver lines generated by GENSAT greatly expands the range of cell type markers that can be used for live cell sorting. In addition, the GENSAT project has generated 278 new Cre driver lines. This review provides an overview of the GENSAT lines and information for identifying lines that may be useful for a particular application. I also provide a review of the few published cases in which GENSAT mice have been used for studies of embryonic development or analysis of stem/progenitor cells in nonneural tissues. genesis 54:245-256, 2016. © 2016 Wiley Periodicals, Inc.

A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish

Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.

Classic cadherin expressions balance postnatal neuronal positioning and dendrite dynamics to elaborate the specific cytoarchitecture of the mouse cortical area

A unique feature of the mammalian cerebral cortex is in its tangential parcellation via anatomical and functional differences. However, the cellular and/or molecular machinery involved in cortical arealization remain largely unknown. Here we map expression profiles of classic cadherins in the postnatal mouse barrel field of the primary somatosensory area (S1BF) and generate a novel bacterial artificial chromosome transgenic (BAC-Tg) mouse line selectively illuminating nuclei of cadherin-6 (Cdh6)-expressing layer IV barrel neurons to confirm that tangential cellular assemblage of S1BF is established by postnatal day 5 (P5). When we electroporate the cadherins expressed in both barrel neurons and thalamo-cortical axon (TCA) terminals limited to the postnatal layer IV neurons, S1BF cytoarchitecture is disorganized with excess elongation of dendrites at P7. Upon delivery of dominant negative molecules for all classic cadherins, tangential cellular positioning and biased dendritic arborization of barrel neurons are significantly altered. These results underscore the value of classic cadherin-mediated sorting among neuronal cell bodies, dendrites and TCA terminals in postnatally elaborating the S1BF-specific tangential cytoarchitecture. Additionally, how the “protocortex” machinery affects classic cadherin expression profiles in the process of cortical arealization is examined and discussed.

Adenine Nucleotide Translocase 4 Is Expressed Within Embryonic Ovaries and Dispensable During Oogenesis

Adenine nucleotide translocase (Ant) facilitates the exchange of adenosine triphosphate across the mitochondrial inner membrane and plays a critical role for bioenergetics in eukaryotes. Mice have 3 Ant paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5), and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. We previously identified that Ant4 was expressed exclusively in testicular germ cells in adult mice and essential for spermatogenesis and subsequently male fertility. Further investigation into the process of spermatogenesis revealed that Ant4 was particularly highly expressed during meiotic prophase I and indispensable for normal progression of leptotene spermatocytes to the stages thereafter. In contrast, the expression and roles of Ant4 in female germ cells have not previously been elucidated. Here, we demonstrate that the Ant4 gene is expressed during embryonic ovarian development during which meiotic prophase I occurs. We confirmed embryonic ovary-specific Ant4 expression using a bacterial artificial chromosome transgene. In contrast to male, however, Ant4 null female mice were fertile although the litter size was slightly decreased. They showed apparently normal ovarian development which was morphologically indistinguishable from the control animals. These data indicate that Ant4 is a meiosis-specific gene expressed during both male and female gametogenesis however indispensable only during spermatogenesis and not oogenesis. The differential effects of Ant4 depletion within the processes of male and female gametogenesis may be explained by meiosis-specific inactivation of the X-linked Ant2 gene in male, a somatic paralog of the Ant4 gene.

Generating a transgenic mouse line stably expressing human MHC surface antigen from a HAC carrying multiple genomic BACs.

The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-014-0488-3) contains supplementary material, which is available to authorized users.

The cross-priming capacity and direct presentation potential of an autoantigen are separable and inversely related properties.

We investigated whether a prevalent epitope of the β-cell-specific autoantigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP206-214) reaches regional Ag-presentation pathways via unprocessed polypeptide chains, as free IGRP206-214 peptide or via preformed IGRP206-214/K(d) complexes. This was accomplished by expressing bacterial artificial chromosome transgenes encoding wild-type (stable) or ubiquitinated (unstable) forms of IGRP in IGRP-deficient NOD mice carrying MHC class I-deficient β-cells, dendritic cells, or B cells. We investigated the ability of the pancreatic lymph nodes of these mice to prime naive IGRP206-214-reactive CD8(+) T cells in vivo, either in response to spontaneous Ag shedding, or to synchronized forms of β-cell necrosis or apoptosis. When IGRP was made unstable by targeting it for proteasomal degradation within β-cells, the cross-priming, autoimmune-initiating potential of this autoantigen (designated autoantigenicity) was impaired. Yet at the same time, the direct presentation, CTL-targeting potential of IGRP (designated pathogenicity) was enhanced. The appearance of IGRP206-214 in regional Ag-presentation pathways was dissociated from transfer of IGRP206-214 or IGRP206-214/K(d) from β cells to dendritic cells. These results indicate that autoantigenicity and pathogenicity are separable and inversely related properties and suggest that pathogenic autoantigens, capable of efficiently priming CTLs while marking target cells for CTL-induced killing, may have a critical balance of these two properties.

A mouse renin distal enhancer is essential for blood pressure homeostasis in BAC-rescued renin-null mutant mice

Renin is predominantly expressed in juxtaglomerular cells in the kidney and regulates blood pressure homeostasis. To examine possible in vivo functions of a mouse distal enhancer (mdE), we generated transgenic mice (TgM) carrying either wild-type or mdE-deficient renin BACs (bacterial artificial chromosome), integrated at the identical chromosomal site. In the kidneys of the TgM, the mdE contributed 80% to basal renin promoter activity. To test for possible physiological roles for the mdE, renin BAC transgenes were used to rescue the hypotensive renin-null mice. Interestingly, renal renin expression in the TgBAC:renin-null compound mice was indistinguishable between the wild-type and mutant BAC carriers. Surprisingly, however, the plasma renin activity and angiotensin I concentration in the mdE compound mutant mice were significantly lower than the same parameters in the control mice, and the mutants were consistently hypotensive, demonstrating that blood pressure homeostasis is regulated through transcriptional cis elements controlling renin activity.

Generation of bacterial artificial chromosome (BAC) transgenic mice.

Transgenic mice are among the most helpful tools to study the role of genes in physiological conditions. In this protocol, we describe the generation of bacterial artificial chromosome (BACs) constructs, which are used to express a gene of interest under a particular promoter. BACs as driver of transgenes have the advantage that a characterization of transcriptional control elements is unnecessary and the construct's size usually reduces position effects from random integration. In the following, we firstly explain in detail the amplification of the BAC, the generation of the targeting construct as well as the recombination by ET-cloning, and the analysis of the recombined clones by Southern blot analysis. Finally, we also describe the preparation of the BACs for oocyte injection. In total, the construction of such BAC transgenes needs around 6-8 weeks.

β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications

Increasing evidence points to nuclear compartmentalization as a contributing mechanism for gene regulation, yet mechanisms for compartmentalization remain unclear. In this paper, we use autonomous targeting of bacterial artificial chromosome (BAC) transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs) within an HBB BAC bias a competition between pericentric versus peripheral heterochromatin targeting toward the nuclear periphery, which correlates with increased H3K9me3 across the β-globin gene cluster and locus control region. Targeting to both heterochromatin compartments is dependent on Suv39H-mediated H3K9me3 methylation. In different chromosomal contexts, PTRs confer no targeting, targeting to pericentric heterochromatin, or targeting to the periphery. A combination of fluorescent in situ hybridization, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over hundreds of kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent lamin-associated domain. Our results demonstrate that multiple cis-elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting.

Short- and Long-Term Effects of LRRK2 on Axon and Dendrite Growth

Mutations in leucine-rich repeat kinase 2 (LRRK2) underlie an autosomal-dominant form of Parkinson's disease (PD) that is clinically indistinguishable from idiopathic PD. The function of LRRK2 is not well understood, but it has become widely accepted that LRRK2 levels or its kinase activity, which is increased by the most commonly observed mutation (G2019S), regulate neurite growth. However, growth has not been measured; it is not known whether mean differences in length correspond to altered rates of growth or retraction, whether axons or dendrites are impacted differentially or whether effects observed are transient or sustained. To address these questions, we compared several developmental milestones in neurons cultured from mice expressing bacterial artificial chromosome transgenes encoding mouse wildtype-LRRK2 or mutant LRRK2-G2019S, Lrrk2 knockout mice and non-transgenic mice. Over the course of three weeks of development on laminin, the data show a sustained, negative effect of LRRK2-G2019S on dendritic growth and arborization, but counter to expectation, dendrites from Lrrk2 knockout mice do not elaborate more rapidly. In contrast, young neurons cultured on a slower growth substrate, poly-L-lysine, show significantly reduced axonal and dendritic motility in Lrrk2 transgenic neurons and significantly increased motility in Lrrk2 knockout neurons with no significant changes in length. Our findings support that LRRK2 can regulate patterns of axonal and dendritic growth, but they also show that effects vary depending on growth substrate and stage of development. Such predictable changes in motility can be exploited in LRRK2 bioassays and guide exploration of LRRK2 function in vivo.

A Bacterial Artificial Chromosome Transgene with PolymorphicCd72Inhibits the Development of Glomerulonephritis and Vasculitis in MRL-FaslprLupus Mice

Systemic lupus erythematosus is considered to be under the control of polygenic inheritance, developing according to the cumulative effects of susceptibility genes with polymorphic alleles; however, the mechanisms underlying the roles of polygenes based on functional and pathological genomics remain uncharacterized. In this study, we substantiate that a CD72 polymorphism in the membrane-distal extracellular domain impacts on both the development of glomerulonephritis and vasculitis in a lupus model strain of mice, MRL/MpJ-Fas(lpr), and the reactivity of BCR signal stimulation. We generated mice carrying a bacterial artificial chromosome transgene originating from C57BL/6 (B6) mice that contains the Cd72(b) locus (Cd72(B6) transgenic [tg]) or the modified Cd72(b) locus with an MRL-derived Cd72(c) allele at the polymorphic region corresponding to the membrane-distal extracellular domain (Cd72(B6/MRL) tg). Cd72(B6) tg mice, but not Cd72(B6/MRL) tg mice, showed a significant reduction in mortality following a marked improvement of disease associated with decreased serum levels of IgG3 and anti-dsDNA Abs. The number of splenic CD4(-)CD8(-) T cells in Cd72(B6) tg mice was decreased significantly in association with a reduced response to B cell receptor signaling. These results indicate that the Cd72 polymorphism affects susceptibility to lupus phenotypes and that novel functional rescue by a bacterial artificial chromosome transgenesis is an efficient approach with wide applications for conducting a genomic analysis of polygene diseases.

A genomic toolkit to investigate kinesin and myosin motor function in cells

Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein-protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.

Transposon mediated BAC transgenesis via pronuclear injection of mouse zygotes

Pronuclear microinjection of bacterial artificial chromosomes (BACs) is the preferred way to generate transgenic mice because the transgene accurately recapitulates expression of the endogenous gene. However, the method is demanding and the integrity and copy number of the BAC transgene is difficult to control. Here, we describe a simpler pronuclear injection method that relies on transposition to introduce full-length BACs into the mouse genome. The bacterial backbone of a hPAX6-GFP reporter BAC was retrofitted with PiggyBac transposon inverted terminal repeats and co-injected with PiggyBac transposase mRNA. Both the frequency of transgenic founders as well as intact, full-length, single copy integrations were increased. Transposition was determined by a rapid PCR screen for a transpositional signature and confirmation by splinkerette sequencing to show that the BACs were integrated as a single copy either in one or two different genomic sites. BAC transposons displayed improved functional accuracy over random integrants as evaluated by expression of the hPAX6-GFP reporter in embryonic neural tube and absence of ectopic expression. This method involves less work to achieve increased frequencies of both transgenesis and single copy, full-length integrations. These advantages are not only relevant to rodents but also for transgenesis in all systems.

Rapid Transcription Fosters Coordinate snail Expression in the Drosophila Embryo

Transcription is commonly held to be a highly stochastic process, resulting in considerable heterogeneity of gene expression among the different cells in a population. Here, we employ quantitative in situ hybridization methods coupled with high-resolution imaging assays to measure the expression of snail, a developmental patterning gene necessary for coordinating the invagination of the mesoderm during gastrulation of the Drosophila embryo. Our measurements of steady-state mRNAs suggest that there is very little variation in snail expression across the different cells that make up the mesoderm and that synthesis approaches the kinetic limits of Pol II processivity. We propose that rapid transcription kinetics and negative autoregulation are responsible for the remarkable homogeneity of snail expression and the coordination of mesoderm invagination.

Disparate SEC23B Deficient Phenotypes in Humans and Mice

Abstract 974SEC23 is a core component of the COPII coated vesicle that mediates the transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. Mutations in human SEC23B, one of two mammalian SEC23 paralogs, results in the autosomal recessive disease, congenital dyserythropoietic anemia type II (CDAII). In contrast, SEC23B deficient mice were recently reported to die perinatally with massive pancreatic degeneration, with no evidence of anemia at birth (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9.) To examine the impact of SEC23B deficiency on hematopoietic function in mice surviving beyond the immediate perinatal period, lethally irradiated C57BL/6J mice were transplanted with fetal liver cells (FLC) collected from E17.5 embryos that were either wild-type (WT) or homozygous for the previously reported Sec23b gene trap allele (Sec23bgt/gt). Recipients of Sec23bgt/gt FLC were indistinguishable from their WT transplant controls and exhibited none of the phenotypic characteristics of CDAII (anemia, increased bi/multi-nucleated RBC precursors, narrower band size and increased shift of the membrane protein band 3 on SDS gel electrophoresis, or RBC double membrane appearance by electron microscopy). To test for a more subtle defect in hematopoietic reconstitution, Sec23bgt/gt FLCs were co-transplanted with WT GFP+ FLC in a 1:1 ratio, with no competitive difference observed over the course of 18 weeks of follow-up. Transplant of marrow from these chimeric animals into secondary recipients demonstrated continued equivalence of Sec23bgt/gt and WT hematopoietic stem cells. To rule out an incidental mutation in a nearby gene (“passenger gene”) as the cause of the pancreatic phenotype in SEC23B-deficient mice, two Sec23b bacterial artificial chromosome transgenes were crossed into the Sec23bgt line, with both demonstrating complete rescue of the Sec23bgt/gt pancreatic phenotype, with normal survival to adulthood. Sec23bgt/gt murine embryonal fibroblasts express a SEC23B/βGEO fusion protein consistent with the gene trap insertion into Sec23b intron 19, and this fusion protein co-immunoprecipitates with SEC24A (Tao, et al., 2012. Proc.Natl.Acad.Sci.U.S.A. 109:E2001–9). To rule out a contribution of the SEC23B/βGEO fusion protein to the disparate human and mouse SEC23B-deficient phenotypes, a second, conditional SEC23B allele was analyzed, in which exons 5 and 6 are flanked by loxP sites (Sec23bfl). Deletion of exons 5 and 6 results in frame shift and stop codon in exon 7. Mice with an erythroid-specific deficiency of SEC23B were generated by crossing the Sec23bfl allele to an EpoR-Cre transgene. Sec23bfl/-/EpoR-CreTg+ mice exhibit no anemia compared to their WT litter mates. Transplant recipients of FLCs from E16.5 embryos homozygous for a germline deletion of Sec23b exons 5 and 6 (Sec23b−/−) were also indistinguishable from mice receiving WT FLCs. Pancreas-specific knock-out generated by crossing the Sec23bfl allele to a p48-Cre or Pdx1-Cre transgene confirmed the pancreatic phenotype observed. This suggests that the perinatal lethality in Sec23b deficient mice may be the result of the loss of pancreatic Sec23b expression. To explore the mechanism for the disparate human and mouse SEC23B-deficient phenotypes, the expression patterns for SEC23A and SEC23B were examined in mice and humans by RT PCR and western blotting. The ratio of SEC23B/SEC23A expression is higher in mouse pancreas (12.7) compared to bone marrow (2.6), whereas in humans, the ratio is higher in the bone marrow (7.8) relative to pancreas (5.5) (normalized to liver). Taken together with the high degree of sequence similarity between SEC23A and SEC23B (∼ 85% identity at the amino acid level), we hypothesize that these 2 SEC23 paralogs overlap extensively at the level of protein function, with the disparate deficiency phenotypes due primarily to differences in tissue and developmentally-specific gene expression programs that have shifted extensively during recent mammalian evolution. These findings have important implications for the comparative function of other closely related paralogous genes. Further studies of the overlapping functions of SEC23A and SEC23B and their relevant protein cargos should provide new insight into the pathogenesis of CDAII and potential therapeutic approaches. Disclosures:Ginsburg:Shire Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Portola Pharmaceuticals: Consultancy; Catalyst Biosciences: Consultancy; Baxter Pharmaceuticals: benefit from payments to Children’s Hosptial, Boston, and the University of Michigan Patents & Royalties; Merck Pharmaceuticals: Consultancy.

Creation and characterization of BAC-transgenic mice with physiological overexpression of epitope-tagged RCAN1 (DSCR1)

The chromosome 21 gene RCAN1, encoding a modulator of the calcineurin (CaN) phosphatase, is a candidate gene for contributing to cognitive disability in people with Down syndrome (DS; trisomy 21). To develop a physiologically relevant model for studying the biochemistry of RCAN1 and its contribution to DS, we generated bacterial artificial chromosome-transgenic (BAC-Tg) mouse lines containing the human RCAN1 gene with a C-terminal HA-FLAG epitope tag incorporated by recombineering. The BAC-Tg was expressed at levels only moderately higher than the native Rcan1 gene: approximately 1.5-fold in RCAN1 (BAC-Tg1) and twofold in RCAN1 (BAC-Tg2). Affinity purification of the RCAN1 protein complex from brains of these mice revealed a core complex of RCAN1 with CaN, glycogen synthase kinase 3-beta (Gsk3b), and calmodulin, with substoichiometric components, including LOC73419. The BAC-Tg mice are fully viable, but long-term synaptic potentiation is impaired in proportion to BAC-Tg dosage in hippocampal brain slices from these mice. RCAN1 can act as a tumor suppressor in some systems, but we found that the RCAN1 BAC-Tg did not reduce mammary cancer growth when present at a low copy number in Tp53;WAP-Cre mice. This work establishes a useful mouse model for investigating the biochemistry and dose-dependent functions of the RCAN1 protein in vivo.

Abstract 38: Rbp-j Regulates the Genetic Program of the Myo-epithelioid JG Cell

RBP-J , the major downstream effector of Notch signaling, is necessary to maintain the number of juxtaglomerular (JG) cells. In addition, RBP-J regulates the plasticity of arteriolar smooth muscle cells to adopt the renin cell phenotype when homeostasis is threatened. We hypothesized that RBP-J acts as an on/off switch controlling the expression of genes that determine the renin phenotype. To determine whether RBP-J directly affects renin gene expression, we generated mice harboring a bacterial artificial chromosome (BAC) transgene with green fluorescent protein (GFP) under the control of the renin gene carrying a mutation in its RBP-J- binding site (Mut-BAC). Mut-BAC mice had markedly reduced GFP expression to 12.9 % ±0.01 (n=3) of the control (Wt-BAC) and a diminished response to homeostatic challenges: mut-BAC mice had a reduced number of GFP positive JG areas per total number of glomeruli (Wt-BAC: 25.1 % ±3.0, n=3; Mut-BAC: 9.3 % ±1.4, n=2, p<0.02) and no GFP expression along the arterioles. To determine whether the decrease in the number of JG cells in mice lacking RBP-J (cKO) was due to a diminished endowment of renin progenitor cells, we traced the fate of cells derived from the renin lineage by generating mice ( RBP-J fl/fl ; Ren1d +/cre ; R26R +/- ) in which cells lacking RBP-J simultaneously expressed β-galactosidase (β-gal). The pattern of β-gal in cKO and control kidneys was identical, indicating that cells derived from the renin lineage did not die but instead changed their phenotype. Next we investigated the phenotype adopted by the cells derived from the renin lineage. Expression of α-smooth muscle actin and smoothelin (a marker of mature smooth muscle) was significantly decreased to 41 % ±7.0 (n=2) and 44 % ±8.8 (n=2) respectively with respect to controls (p<0.01). In addition, mutant JG cells in vivo did not express genes characteristic of the renin phenotype such as renin, calponin1, Nfat and Akr1b7 expressing instead fibroblast-specific protein 1 indicating the adoption of a fibroblast-like phenotype. Results indicate that RBP-J directly governs a genetic program that controls the dual endocrine-contractile phenotype of the JG cell, which is crucial to maintain blood pressure and fluid-electrolyte homeostasis.