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      The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

      research-article
      a , 1 , a , a , a , b , a , a , *
      Molecular and Cellular Neurosciences
      Academic Press
      AOTF, acousto-optic tunable filter, BAC, bacterial artificial chromosome, CDS, coding sequence, CMV, cytomegalovirus, DOR, δ-opioid receptor, DR, dorsal raphe nucleus, DRG, dorsal root ganglia, EGFP, enhanced green fluorescent protein, ER, endoplasmic reticulum, ES cell, embryonic stem cell, FRT sites, FLP recombination target sites, GALP, galanin-like peptide, Gapdh, glyceraldehyde 3-phosphate dehydrogenase, GFP, green fluorescent protein, GPCRs, G protein-coupled receptors, hrGFP, humanized Renilla green fluorescent protein, ISH, in situ hybridization, LSN, lateral spinal nucleus, ME, median eminence, NPY, neuropeptide Y, nt, nucleotides, RNA-seq, next generation RNA sequencing, RT-PCR, reverse transcription polymerase chain reaction, TSA, tyramide signal amplification, UTR, untranslated region, uORFs, upstream open reading frames, GalR1, GalR2, Dorsal root ganglion, Spinal cord, Brain, uORF

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          Abstract

          The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal 1, Gal 2 and the less studied Gal 3 ( GalR13 gene products). There is a wealth of data on expression of Gal 1–3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal 1 or Gal 2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal 1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal 1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal 1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal 2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal 1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs).

          Highlights

          • We generated knock-in mice expressing fluorescently tagged galanin receptors 1 and 2.

          • Gal 1-mCherry fluorescence was associated with primary neuron somatic cell membrane.

          • Gal 1-mCherry protein expression in DRG, spinal cord and brain tissues is described.

          • Gal 2 mRNA expression is much lower than Gal 1 in dorsal root ganglia.

          • We propose that upstream open reading frames influence Gal 2 protein expression.

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          Most cited references119

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          Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans.

          Upstream ORFs (uORFs) are mRNA elements defined by a start codon in the 5' UTR that is out-of-frame with the main coding sequence. Although uORFs are present in approximately half of human and mouse transcripts, no study has investigated their global impact on protein expression. Here, we report that uORFs correlate with significantly reduced protein expression of the downstream ORF, based on analysis of 11,649 matched mRNA and protein measurements from 4 published mammalian studies. Using reporter constructs to test 25 selected uORFs, we estimate that uORFs typically reduce protein expression by 30-80%, with a modest impact on mRNA levels. We additionally identify polymorphisms that alter uORF presence in 509 human genes. Finally, we report that 5 uORF-altering mutations, detected within genes previously linked to human diseases, dramatically silence expression of the downstream protein. Together, our results suggest that uORFs influence the protein expression of thousands of mammalian genes and that variation in these elements can influence human phenotype and disease.
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            A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.

            Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective lambda prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era. Copyright 2001 Academic Press.
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              HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells.

              Embryonal stem (ES) cell lines, established in culture from peri-implantation mouse blastocysts, can colonize both the somatic and germ-cell lineages of chimaeric mice following injection into host blastocysts. Recently, ES cells with multiple integrations of retroviral sequences have been used to introduce these sequences into the germ-line of chimaeric mice, demonstrating an alternative to the microinjection of fertilized eggs for the production of transgenic mice. However, the properties of ES cells raise a unique possibility: that of using the techniques of somatic cell genetics to select cells with genetic modifications such as recessive mutations, and of introducing these mutations into the mouse germ line. Here we report the realization of this possibility by the selection in vitro of variant ES cells deficient in hypoxanthine guanine phosphoribosyl transferase (HPRT; EC 2.4.2.8), their use to produce germline chimaeras resulting in female offspring heterozygous for HPRT-deficiency, and the generation of HPRT-deficient preimplantation embryos from these females. In human males, HPRT deficiency causes Lesch-Nyhan syndrome, which is characterized by mental retardation and self-mutilation.
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                Author and article information

                Contributors
                Journal
                Mol Cell Neurosci
                Mol. Cell. Neurosci
                Molecular and Cellular Neurosciences
                Academic Press
                1044-7431
                1095-9327
                1 September 2015
                September 2015
                : 68
                : 258-271
                Affiliations
                [a ]Schools of Physiology and Pharmacology and Clinical Sciences, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK
                [b ]Wolfson Bioimaging Facility, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK
                Author notes
                [* ]Corresponding author. d.wynick@ 123456bristol.ac.uk
                [1]

                Current address: MRC Harwell, Didcot, Oxfordshire OX11 0RD, UK.

                Article
                S1044-7431(15)30012-9
                10.1016/j.mcn.2015.08.006
                4604734
                26292267
                4db9282d-502f-4b43-961a-838b9afdbfd2
                © university of bristol. Published by Elsevier Inc.

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 20 May 2015
                : 6 August 2015
                : 10 August 2015
                Categories
                Article

                Neurosciences
                aotf, acousto-optic tunable filter,bac, bacterial artificial chromosome,cds, coding sequence,cmv, cytomegalovirus,dor, δ-opioid receptor,dr, dorsal raphe nucleus,drg, dorsal root ganglia,egfp, enhanced green fluorescent protein,er, endoplasmic reticulum,es cell, embryonic stem cell,frt sites, flp recombination target sites,galp, galanin-like peptide,gapdh, glyceraldehyde 3-phosphate dehydrogenase,gfp, green fluorescent protein,gpcrs, g protein-coupled receptors,hrgfp, humanized renilla green fluorescent protein,ish, in situ hybridization,lsn, lateral spinal nucleus,me, median eminence,npy, neuropeptide y,nt, nucleotides,rna-seq, next generation rna sequencing,rt-pcr, reverse transcription polymerase chain reaction,tsa, tyramide signal amplification,utr, untranslated region,uorfs, upstream open reading frames,galr1,galr2,dorsal root ganglion,spinal cord,brain,uorf

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