Transgenic mice expressing yellow fluorescent protein under control of the human tyrosine hydroxylase promoter
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Transgenic Mice Expressing YellowFluorescent Protein Under Control of theHuman Tyrosine Hydroxylase Promoter
Eun Yang Choi,1 Jae Won Yang,1 Myung Sun Park,1 Woong Sun,2
Hyun Kim,2 Seung U. Kim,2,3 and Myung Ae Lee1*1Brain Disease Research Center, and Institute for Medical Sciences, Ajou University School of Medicine,Suwon, Korea2Department of Anatomy, Korea University College of Medicine, Seoul, Korea3Medical Research Institute, Chung-Ang University School of Medicine, Seoul, Korea4Division of Neurology, Department of Medicine, University of British Columbia, Vancouver,British Columbia, Canada
Pathogenesis of Parkinsons disease and related cate-cholaminergic neurological disorders is closely associ-ated with changes in the levels of tyrosine hydroxylase(TH). Therefore, investigation of the regulation of the THgene system should assist in understanding the patho-mechanisms involved in these neurological disorders. Toidentify regulatory domains that direct human TH expres-sion in the central nervous system (CNS), we generatedtwo transgenic mouse lines in which enhanced yellowuorescent protein (EYFP) is expressed under the controlof either 3.2-kb (hTHP-EYFP construct) human TH pro-moter or 3.2-kb promoter with 2-kb 30-anking regions(hTHP-ex3-EYFP construct) of the TH gene. In the adulttransgenic mouse brain, the hTHP-EYFP constructdirects neuron-specic EYFP expression in various CNSareas, such as olfactory bulb, striatum, interpeduncularnucleus, cerebral cortex, hippocampus, and particularlydentate gyrus. Although these EYFP-positive cells wereidentied as mature neurons, few EYFP-positive cellswere TH-positive neurons. On the other hand, we coulddetect the EYFP mRNA expression in a subset of neu-rons in the olfactory bulb, midbrain, and cerebellum, inwhich expression of endogenous TH is enriched, withhTHP-ex3-EYFP transgenic mice. These results indicatethat the 3.2-kb sequence upstream of the TH gene is notsufcient for proper expression and that the 2-kbsequence from the translation start site to exon 3 is nec-essary for expression of EYFP in a subset of catechola-minergic neurons. VVC 2012 Wiley Periodicals, Inc.
Key words: tyrosine hydroxylase; promoter; EYFP;catecholaminergic neuron; transgenic mice
Tyrosine hydroxylase (TH) catalyzes the rate-limit-ing step of hydroxylating tyrosine to dihydroxyphenyla-lanine (DOPA) in the synthesis of catecholamine neuro-transmitters (Nagatsu et al., 1964). In the central nervoussystem (CNS), TH is expressed in dopaminergic(DAergic) neurons of the substantia nigra (SN), ventraltegmentum, hypothalamus, and olfactory bulb; in norad-
renergic neurons of the locus ceruleus and lateral teg-mental system; and in adrenergic neurons of the brain-stem (Zigmond et al., 1989). The mechanisms of THgene expression have been intensively studied, becausecatecholamines play fundamental and important role inneurophysiology and pathogenesis of neurodegenrativediseases, including Parkinsons disease (PD). AberrantTH gene expression is also associated with psychiatricdisorders, such as schizophrenia, bipolar disorder, andside effects caused by alcoholism. More importantly,degeneration and cell death of TH-positive DAergicneurons in the SN are the major cause of PD. A recentstudy has reported that TH alterations and SN neuropa-thology arte also implicated in Huntingtons disease(Yohrling et al., 2003).
We previously reported that a 3.2-kb sequence ofthe human TH gene promoter contains functional pro-moter and cis elements and effectively regulates celltype-specic expression (T.E. Kim et al., 2003). Toidentify regulatory domains that direct human THexpression in the CNS, we generated transgenic mice,hTHP-EYFP, in which enhanced yellow uorescentprotein (EYFP) is expressed under control of the 3.2-kblength of human TH promoters. Earlier studies of THpromoter in transgenic mice have shown that a sequence
Contract grant sponsor: BK21 Program of the Ministry of Education and
Human Resource Development; Contract grant sponsor: KOSEF/BDRC
Ajou University from the Korean Ministry of Science and Technology;
Contract grant sponsor: Neurobiology Research Program grant from the
Korean Ministry of Science and Technology; Contract grant sponsor: Stem
Cell Research Center of the 21st Century Frontier Research Program
(SC3090) from the KoreanMinistry of Science and Technology.
*Correspondence to: Myung Ae Lee, PhD, Brain Disease Research
Center, and Institute for Medical Sciences, Ajou University School of
Medicine, Suwon, Korea 442-749. E-mail: email@example.com
Received 22 March 2012; Accepted 15 April 2012
Published online in Wiley Online Library (wileyonlinelibrary.com).
Journal of Neuroscience Research 00:000000 (2012)
' 2012 Wiley Periodicals, Inc.
of 511 kb is required for high-level expression of thereporter in catecholamine neurons (Sasaoka et al., 1992;Min et al., 1994; Liu et al., 1997; Trocme et al., 1998;Sawamoto et al., 2001; Matsushita et al., 2002; Kessleret al., 2003). Other studies have demonstrated that thehuman TH promoter of 2.5 kb, including the entireexonintron structure with 0.5 kb of the 30-ankingregion, is sufcient for tissue-specic expression of TH intransgenic mice (Kaneda et al., 1991). Therefore, toinvestigate further the role of hTH gene in tissue-specicexpression, we generated an additional transgenic mouseline, hTHP-ex3-EYFP, which contains the sameupstream region as hTHP-EYFP, and 2-kb sequencefrom the translation start site to exon 3. Our currentobservations suggest that the 3.2-kb sequence upstream ofthe TH gene is not sufcient for proper expression andsuggest the importance of the 2-kb sequence from thetranslation start site to exon 3 for the expression of down-stream genes in a subset of catecholaminergic neurons.
MATERIALS AND METHODS
Construction of Transgenes With EYFP Reporter
Human TH promoter fragment of 3.2 kb produced bySalI-KpnI restriction enzyme digestion of THP4434-pGEM3zf1 was inserted upstream of the EYFP gene inpEYFP plasmid. For normal transcription of the EYFP gene,it was directly connected with the EYFP gene using Quik-Change II site-directed mutagenesis kits (Stratagene, La Jolla,CA). A 0.7-kb fragment of the SV40 poly-A gene frompcDNA3.1/His/lacZ was inserted into the ApoI site down-stream of EYFP to stabilize mRNAs, resulting in the hTHP-EYFP construct. To generate hTHP-ex3-EYFP construct, weconnected a 3.2-kb human TH upstream genomic fragmentand a 2-kb sequence from the translation start site nucleotide76 of exon 3 to the EYFP coding sequence.
Generation and Genotyping of hTH-EYFP TransgenicMice
Transgenic mice were generated by pronuclear microin-jection of fertilized (C57BL/6J 3 DBA/2J) F2 mouse oocytes.To identify founder mice, the genotypes of all offspring wereanalyzed by polymerase chain reaction (PCR). GenomicDNA was prepared from tail biopsies. The thermocycle pro-le for PCR amplication was 1 min at 948C, 1 min at 608C,and 2 min at 728C for 22 cycles to distinguish homozygousmice from heterozygous mice. The primers for PCR analysiswere sense primer for human TH gene, 50-TTTAG-GAAAGGTCCCAGGGG-30; antisense primer for transgene,50-TTGGAGAGACCT TTGCAG TT-30, to yield a 640-bpproduct. The PCR products were separated on a 1.5% agarosegel, stained with ethidium bromide, and quantitatively ana-lyzed in Image Gauge 4.0 (Fuji Film, Tokyo, Japan).
The animals were perfused with 4% paraformaldehyde(PFA) in 0.1 M phosphate buffer, and brains were removedand xed in the same xative at 48C for 16 hr. After washingin phosphate-buffered saline (PBS) for 20 min, the brains were
equilibrated in 30% sucrose in PBS and frozen in dry ice. Thefrozen brains were cut into 30-lm sections with a CM 3000cryostat (Leica Microsystems, Milan, Italy), and the sectionswere permeabilized and blocked with PBS containing 3% goatserum and 0.2% Triton X-100 for 2 hr, then stained with pri-mary antibodies at 48C overnight. After three washes withPBS, the sections were incubated for 1 hr at room temperaturewith Texas red- or Cy3-conjugated secondary antibodies(1:200500; Vector, Burlingame, CA) in PBS containing 3%bovine serum albumin. The sections were then mounted on aglass slide with PermaFluor (Thermo Shandon, Pittsburgh,PA). Fluorescence images were obtained under a confocal laserscanning microscope (Olympus, Tokyo, Japan). Primary anti-bodies used were TH (1:500, sheep; Pel-Freeze, Rogers, AR),NeuN (1: 400, mouse mAb; Chemicon, Temecula, CA), dou-blecortin (DCX; 1:500, rabbit; Chemicon), calretinin (CR;1:500, rabbit; Chemicon), and calbindin (CB; 1:500, mousemAb; Chemicon). For immunodetection of EYFP, we usedanti-GFP antibody (1:100, rabbit; BD Bioscience, San Jose,CA) and biotinylated anti-rabbit antibody (1:200; Vector), anABC kit, and a diaminobenzidine (DAB) staining kit (Vector).
Total RNA was extracted from various tissues of mousebrain using RNeasy mini kit (Qiagen, Valencia, CA). Twomicrograms of RNA was reverse transcribed with SuperscriptII Reverse Transcriptase (Invitrogen, Carlsbad, CA) in thepresence of random primers, according to the manufacturersinstructions. The resulting cDNA was amplied by PCRusing primers specic for human TH and EYFP. Primerswere TH forward 50-CTGAGCCATGCCCACCCCC-GACGCCACC AC-30 and two EYFP reverse 50-TGAA-GAAGATGGTGC GCTCCTGGAC-30 and 50-GGTTCAC-CAGGGTGTCGC CC-30. Amplication was performed in aPTC-200 thermal cycler (MJ Research, Toronto, Ontario,Canada), and conditions were 30 cycles of 1 min at 948C, 1min at 658C, and 2 min at 728C. Amplied products wereseparated on a 2% agarose gel containing ethidium bromide.
Southern Blot Analysis
PCRs were performed using cDNAs from brain tissuesof hTHP-ex3-EYFP transgenic mice. PCR products wereresolved on a 1.2% agarose gel and transferred to a positivelycharged nylon membrane (Roche Diagnostics, Indianapolis,IN). The blot was hybridized overnight at 428C in hybridiza-tion buffer with a human TH-gene-derived 400-bp XbaIprobe that had been labeled with 32P-dCTP (3,000 Ci/mmol)by DNA polymerase extension of random hexamers (Boeh-ringer Mannheim, Indianapolis, IN).
In Situ Hybridization
Mouse TH cDNA (nucleotides 1801382 of GenBankaccession No. BC053706) was obtained from the KoreanGenBank and EYFP cDNA from pEYFP plasmid (Promega,Madison, WI). These cDNAs were cloned into the pGEMT-easy plasmid (Promega) and used for riboprobe synthesis.Sections from wild-type and transgenic brains (coronal andsagittal, 12 lm thick) were collected in serial order using a
2 Choi et al.
Journal of Neuroscience Research
cryostat (Leica), xed in 4% paraformaldehyde (PFA), andhybridized with radiolabeled riboprobes. Hybridization andwashing conditions were described previously (Kim et al.,1994). For autoradiography, the slides were exposed at 2208Cto b-Max lm (Amersham, Arlington Heights, IL) for 4 days.For darkeld and brighteld microscopy, the slides were dippedin NTB2 nuclear track emulsion (Eastman Kodak, Rochester,NY) and incubated at 48C for 1 week; sections were thendeveloped, lightly counterstained with cresyl violet (FisherScientic, Seoul, Korea), and overlaid with coverslips.
Generation of Human TH-EYFP Transgenic Mice
To determine whether the TH upstream sequencedirects its expression in a cell-specic and developmen-tally regulated manner, we fused the 50-anking regionwith or without the rst three exons of the human THgene to the EYFP reporter gene (hTHP-ex3-EYFP andhTHP-EYFP constructs, respectively; Fig. 1A). For thehTHP-EYFP construct, the fusion gene consists of a3.2-kb hTH upstream genomic fragment (starting at thehTH translation initiation site), the EYFP gene, and apolyadenylation site derived from the SV40 gene (Fig.
1A). For the hTHP-ex3-EYFP construct, we used a3.2-kb hTH upstream genomic fragment and a 2-kbsequence from the translation start site to exon 3(Fig. 1A).
We have generated transgenic mice, using thesetwo constructs, with the aim of identifying a region (orregions) that transactivates TH transcription specicallyin the brain. PCR analyses of tail DNAs identied veand three independent transgenic lines that passed theirtransgenes onto their offspring for the hTHP-EYFP andthe hTHP-ex3-EYFP constructs, respectively (Fig. 1B).Three of ve hTHP-EYFP lines (named hTHP-EYFP7,-9, and -131) and three of hTHP-ex3-EYFP linesshowed mRNA expression in the brain (data notshown). To assess the tissue specicity of EYFP expres-sion, we tested the EYFP mRNA expression by RT-PCR (Fig. 1C). EYFP mRNA was expressed strongly inthe hippocampus, cerebral cortex, and olfactory bulb(OB) and only weakly in the midbrain of hTHP-EYFPtransgenic mice. By contrast, EYFP mRNA was ratherubiquitously observed in most brain regions of hTHP-ex3-EYFP mice. These brain-region-dependent expres-sion patterns are distinct from the distribution of endog-enous TH mRNA. When tissue specicity of EYFP
Fig. 1. Human TH-EYFP transgenic mice. A: Schematic of thehuman TH-EYFP and TH-ex3-EYFP transgene constructs. The 3.2-kb SalI-Eco52I fragment from the 50-anking region of human THgene was fused with EYFP cDNA (yellow box) and a polyadenyl-ation sequence to generate the TH-EYFP construct. The TH-ex3-EYFP construct was generated to analyze the function of genomicsequences other than the 50-anking region on TH gene expressionin vivo and includes the region from 23174 to exon 3 of the THgene, the EYFP reporter gene, and polyadenylation signals. B: PCR-
based genotype screening for the generation of transgenic mice. The640-bp band corresponds to the human TH gene. DW, distilledwater; WT, wild type; PC, positive control. C: RT-PCR analysis ofhuman TH promoter-driven mRNA expression from various mousebrain regions of transgenic mice. HC, hippocampus; Ctx, cerebralcortex; MB, midbrain; OB, olfactory bulb; St, striatum; VTA, ventraltegmental area; CB, cerebellum cortex; LC, locus ceruleus; M, DNAsize marker. [Color gure can be viewed in the online issue, whichis available at wileyonlinelibrary.com.]
EYFP Expression Driven by Human TH Promoter 3
Journal of Neuroscience Research
expression was assessed via uorescence microscopy,strong EYFP uorescence was found in several brainregions of hTHP-EYFP mice.
Characterization of hTHP-EYFP Transgenic Mice
Next we addressed the cellular-level expression ofEYFP signals in brain tissue sections of hTHP-EYFPtransgenic mice by epiuorescence or laser scanningconfocal microscopy (Fig. 2). Because the hTHP-EYFP7line showed the strongest EYFP uorescence signals, allexperiments were performed with this transgenic mouseline. Strong EYFP staining was seen in the anterior ol-factory nucleus, olfactory granule cell layers, CA13region, and dentate gyrus (DG) of the hippocampus, cer-ebellum, and cerebral cortex, whereas no staining wasfound in the globus pallidus or substantia nigra (SN; Fig.2). On the other hand, the slices prepared from the non-transgenic mice showed no signals (data not shown).Expression of EYFP in the adult brain is summarized inTable I.