transgenic mice expressing yellow fluorescent protein under control of the human tyrosine...
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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: firstname.lastname@example.org
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 g