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Tgp chi Cong nghe Sinh hoc 10(3): 407-413, 2012 TAO DONG RUOI GIAM CHUYEN GEN MANG PHITC HOP UAS-DUCH DE NGHIEN CIYU VAI TR6 CUA PROTEIN UCH-L1 DOI YOI BENH PARKINSON Phan Nguyen Thyy An', D4ng Thi Phtrung Than% Masamistu Yamaguchi2, Tan Linh Think' I Trzrang Dgi hoc Khoa hoc to nhien, Dgi hoc Quac Gia Thanh pha Ho Chi Minh Cong nghe Kyoto, Nhdt Bcin Vien TOM TAT Benh Pakinson la benh thoai hoa he than kinh van,d6ng ph6 Wen nguiri cao tuoi, nguyen nhan la nao ix) nguai benh khong sin xuat dtrqc chat dan truyen than kinh dopamine, an den mat 'chi rang kiem soat hoat Clang van clang ca Nhting nghien ciru gan day cung cap nhieu bang chimg sinh hoc phan ti lien quan den, CC/ thOng a-synuclein,.su thily phan chuoi ubiquitin the phat sinh benh Parkinson nhu sir tich ubiquitin-proteasome bi sai hOng, su mat chic Wang dia ty the va hien tuqng stress oxy hoa Trong de, UCHLI (ubiquitin carboxyl-terminal hydrolase 1) la mat protein 06 Wen nao, co vai tro quan trong he th6ng ubiquitin-proteasome nham phan huy cac protein gap cuan sai to bao Trong nhung nam gan day, mac du nhieu nghien cuu da duqc thuc hien de tim moi quan he eirra UCH-L1 va benh Parkinson ding nhtr cac benh thoai hoa than kinh khac, nhien van de van nhieu tranh cai Be hieu r6 han ye vai tre ctia UCH-L1 ru6i protein UCH-L1, chimg toi.tien hinh bieu hien protein dUCH, mat protein tuang giam, tong ru'Oi giam chuyen gen va nghien ciru vai to can no Bang each tao vector pUAST-dUCH, vi tiem vao ru6i giAm nham tao cac dong mai giam chuyen gen mang phirc hop UAS-dUCH va bieu hien dinh huang dUCH tai cac mo dich thong qua phirc hqp GAL4-UAS Khi lai cac clang UAS-dUCH vai clang GMRGAL4, dUCH duqc bieu hien dinh htrang a m8 mat, phan tich cho thay protein dUCH bieu hien manh a vimg phan bao thir cap co vai tro quan trong qua trinh hinh mat va a \rung duoi cua dia tien plan sinh mat chimg to ce mei lien he gifra mire d6 bieu hien dUCH va kieu hinh mat ruoi Nhu vay, ruoi giam chuyen gen dUCH IA met mo hinh tot de co the nghien ciru vai to dm protein doi vai benh Parkinson ye sau Tir khoa: gen dUCH, rui5i gam GMR-GAL4, ehuyn gen, bidu hien dinh Intong mo mat GICSI TH1eU Benh Parkinson la met tong nhfing benh ve fel loan than kinh bien nhat, ding sau benh Alzheimer Trong suet the kY 20, yen to di truyen khong ducrc xem la nguyen nhan gay nen benh Parkinson (Corti et aL, 2005): Tuy nhien, nhang nghien cum On day d5 bac be quan diem va quan han nirra la cac nghien ciru da cung cap cac bang ching hieu dtrqc ca the phan dr lien quan den co the phat sinh benh Parkinson (Gasser, 2007) Nghien ciru chic nang va hoat &Ong cea quan cua nhiing1 gen gift') hieu duqc mei met so co the vii va su lien he yea co the phat sinh benh Parkinson to bao, nhu stir tich tu asynuclein, su thity phan chai ubiquitin he thong ubiquitin-proteasome hu hong, str sai chic nang cua ty the va hien tuqng stress oxy hoa (Dawson, Dawson, 2003) Trong do, UCH-L1 la met protein hien dien rat nhieu nao (khoang 1% - 2%) (Wilkinson et aL, 1989), thuec ho enzyme co vai to viec hinh ket i dau C dm protein quy trinh gin ubiquitin cua nhang protein gap cuen sai, protein gia coi khong mong muon can phan hey bii proteasome (Amerik, Hochstrasser, 2004) UCH-L1 tach chuoi ubiquitin khoi protein tnrec protein vao he thong phan cat proteasome, ben canh UCH-L1 phan tach chit& ubiquitin cac monomer ubiquitin, tai sir dung ubiquitin cho he thong ubiquitin-proteasome (Larsen et al., 1998) Mac du ca che gay benh Parkinson van chua duqc biet ro, hau het cac nghien ciru deu quy ye met mei moi rang sir sai h6ng he thong ubiquitinproteasome va sir tich ttu nhimg protein bi bien doi co lien quan den benh Parkinson (Hegde, Upadhya, 2007) Protein UCH-L1 co vai tro trung tam hoat &Ong phan hit)/ protein ciia he thong ubiquitinproteasome nen cac nha nghien cqu cho rang UCHL I ce lien quan den benh Parkinson (Leroy et al., 1998; Maraganore et al., 1999; Nishikawa et al., 2003), nhien van de van nhieu tranh cal Trong nhieu th5p kY gan day, benh di truyen ngtr&i luon la de tai thu het sty quan tam dm cac nha khoa hoc CUng vii si,r phat trien cua k9 thuat sinh 407 Phan Nguyen Thuy An et al hoc phan tir, cat c8ng trinh nghien cirt, ca the sinh hoc phan tir,cita benh duqc ti'en hinh tic mirc de tir in vitro, to bao deng vat cho den in vivo, deng vat chuyen gen Trong so tic m8 hinh &Ong vat chuyen gen, yeti nhang tru diem vuot trei, ruoi giam Drosophila melanogaster la ma hinh duct chQn sir dung bien nghien ciru y - sinh hoc hien Trong nghien ciru nay, than toi xay dung me hinh nghien ciru benh tren Ong vat chuyen gen, ma hinh ruoi giam D melanogaster nham img clung nghien ciru vai tro caa protein UCH-L1 doi voi benh Parkinson Cic ket qui dot duqc bao gam tao thing gen dUCH vaosvector pUAST va chuyen vio ca the ruoi giam nham tao ruoi giam chuyen gen mang phirc hqp UAS-dUCH phut vu cho nghien cuu vai tre dm protein UCH-LI doi \fed benh Parkinson vi xem bieu hien vtayt mirc ciia protein dUCH ruoi ruoi giam chuyen gen VAT LIEU VA PHUONG PHAP Chung vi sinh vat vi vector Chung E coil DH5a (F-endAl hsdR17 (rk-Ank-) supE44 thi X-recAl gyrA96 lacU169 (4)80 lacZ AM15)) dugc sir dung lim the nhAri, nhan va chQn Vector pUAST (Chromosomal Technology Laboratory, Kyoto Institute Technology, Japan) duqc sir dung chuyen gen dUCH -vio ruoi giant Vector pUAST bao gam (loan UAS la trinh to gan protein GAL4, doan polylinker giup viec tao clang duqc de ding vi met trinh to ngimg phi& mA, tat ca duqc dit met vector co the chuyen vi nha nhan to P, co gen Amp` co Ichi nang Ichang Ampicillin giup sang 19c qui trinh tao dang phenol: chloroform, clio Id NIA lac rung Sau ly tam nhu tren, dich , dtr9c bo sung 500 chloroform vi dio ki Sau ly tam nhu ten vi dich not duor be) sung 1/10 the tich M Na acetate pH 5,2 NIA 500 ml 99% ethanol va u 30 phiit -20 C, sau ly tam 30 phut Yeti 13000 vong/phirt 4°C Tim duct Kra lai bang 500p1 70% ethanol, ly 6'11130 phiu vai 13000 vang/phiit 4°C Tim duct de kilo to nhien va giii 50 gl MiliQ Thu nh'On plasmid pUAST-dUCH Plasmid pUAST-dUCH duqc tich chiet tir E coli DH5a theo phuang phial) kiem SDS (Birnboim, Doly, 1979) Khuech dai gen dUCH bang phtrang phip PCR PCR khuech dai gen bao gam ill dem 10x PCR, gl mM dNTP, 5µl 25mM MgCl2, ul DNA be gen (Y ng), gl unit Taq polymerase vi me)i mai (10 pmol), duct b6 sung 31 1.11 ntrac cat len the tich cuoi cang li 50 pl Phan img PCR duct thuc hien theo the de nhiet: 95°C/5', 30 chu kSt (95°C/1', 55°C/1', 72°C/1'), 72°C/10' Thik ke plasmid va bien nap Sal? phim PCR gen dUCH duqc cat vai enzyme han the EcoRI va Xhol, Chen vio vector pUAST tao thinh plasmid tai to hqp pUAST-dUCH va duqc bien nap vio E co/i theo phuang phi!) shock nhiet NIA CaC12 (Sambrook, Russell, 2001) Khung dot cira gen dUCH duqc kiem tra bang PCR, cat hp the va dot trinh tu Tat ca tic thao tic gen deu thuc hien theo phuang phi]) ca bin Sambrook vi Russell (2001) Vi tiem Chung ruoi giim Drosophila melanogaster CantonS va W1118 duqc sir dung nhu clang ruoi hoang dai (Bloomington, My) GMR-Ga14(X) drat dung de bieu hien mot mac gen dUCH a m8 mit Thu nh4n 130 gen ruoi giftm Ruei truing thinh (10 - 15 con) dirge thu vi gift -20 C qua dem: Sau do, hoa 0,1 ml dem ly giii va nghien bang chay, be' sung 0,4 ml dem ly giii H8n hqp duqc bo sung protease K 0,5 mg/ml va gi6 37 C Sau ly tam 10 phut Writ 10000 Yang/phut, 4°C, dich not duqc bo sung 250:250 ill 408 Ruoi dung de chuyen gen phai co hai die diem kh8ng mang gen ma hoa transposase vi khong mang chi thi then 19c (thm:mg ruoi dot bien mat tang) Ru8i duqc nuoi mai truing de - ngiy viz& vi tiem Vio ngiy vi tiem, cu each 60 - 70 phin thay mai truing SY (5% duang, 5% num men, 1% agar) met Ian Tnrac tiem phii de ruoi de trong 20 - 40 phut mai truing thach SY Lop ming diy bao quanh du9c loai be giitp cho qui trinh vi tiem de ding Helper P - element vi plasmid mang nhan to P duqc tiem vio tang met luc a phan duoi cua trimg nai hinh thinh mum to bio Tap chi Cong nghe Sinh hoc 10(3): 407-413, 2012 Trimg ruoi sau vi tiem dugc cho troy lai moi tetrang SY de trUng Oat trien Moi ken ruoi duce nuoi mOt Ong moi trtrang, de chung na ruoi trtremg hoac chi cho den ken no ruoi truong thi bat trinh nu Toan b0 the 114 Fo co matt mat Moi cai Fo dugc giao phoi yen ba duc W 1118 va moi due Fo dugc giao phoi vht ba ,cai WI 11 The 114 F1 c6 kieu hinh sau: Tat ca the h4 Fi co mau mat tang Day la nhimg ruoi khong dugc chuyen gen MOt so ruoi F1 (khoing10%) mat c6 sac to Day la dong ruoi dugc chuyen ,gen The 114 ,F, (han 50%) co nhieu kieu mau mat khac Dieu c6 nghia la co nhieu doan gen da dugc chuyen vao 130 gen ruoi Ruoi co mau mat nhat duce dung de lai tiep theo Sau sang I9c de thu dugc dong ruoi chuyen gen, cac dong ruoi dugc xac dinh nhiem sac the mang gen ellen bang cac phep lai voi ruoi mang chi thi nhiem sac the (balancer) Lai Western MAu protein tren gel di4n di, SDS-PAGE dugc chuygn len mang nitrocellulose bang tac nhan di4n Khea mang dung dich sira gay 5% PBST (80 mM Na,HPO4, 25 mM Nal-14304, 100 mM NaCI, 0,1% Tween 20), lac nhe I gia cho dung dich ti4p Vic deu vori be mat dm mang lai Sau do, ngain mang lai khang the khang dUCH (1/10000) gi6,dong thai lac nhg nhang hop Sau rira ki mang Ian dung dich PBST, moi Ian phin Tiep tuc u mang vai khang the tit& cap danh dau bang HRP pha loang dung dich PBST vai ti 14 1/20000 I gia, dong thai lac nhe hop Sau rim ki mang bang dung dich PBST Ian, mai lin phin Hi4n phim bang dung dich ECL1 va ECL2 (Thermo Fisher Scientific Inc., Waltham, USA) Mien dich hu5,nh quang (Immuno Flourescene) Tach dia tier 'lan sinh mat to Au tang th6 PBS (140 IAM NaCI, ttM KCI, ttM Na2HPO4, va 15 pM KH,PO4 , pH 7,4) wen da CO dinh dia 2% paraformadehydc PBS C qua dem., Sau d6 rira Ian bang PBS nhigt de) phong U vori khang the sa cap a-dUCH antibody (1/10000) C qua dem Rita Ian bang PBS va yeti khang the (ha cap a-FITC antibody (1/1500) gio o ‘nhigt dO phong lioac C qua dem Rim Ian bang PBS va ii mau 100% glycerol 30 phut Xem tin hign bang kinh hien vi huynh quang KET QUA VA BIEN LUAN Thiet ke plasmid tai to hqp pUAST-dUCH San phim gen dUCH vai khuan DNA bO gen mai giam va cap moi dac hign co kich thtrac 980 by dung nhu thiet kg (Hinh 1A, gieng 2) San phArn PCR gen dUCH dugc tinh sach, cat vai EcoRI, Xhol, tinh sach va not dau dinh vao vector pUAST ding dugc cat vai EcoRI va Xhol, bang DNA 8800 by (Hinh 1B, gigng 3) tao plasmid tai to hqp pUASTdUCH Plasmid dugc !Dien nap E coil DI-15a tao cac (long E coil DH5a/pUAST-dUCH Kiem tra khufin lac bang PCR vai cap mo ll wiz] /wiz2, cac khuin lac gieng 4, 5, 8, 9, 10 cho mOt bang DNA — 1030 by nam glib 1000 by va 1500 bp, tuong Ung vai san phi'm PCR plasmid tai to hqp pUAST-dUCH vai cap moi wizl/wiz2., Cie khuan lac mang pUAST-dUCH dugc nuoi cay va tach plasmid bang phucmg pita!) SDS-kiem d4 ki4m tra bang moi dac hi& gen Trong so khuan lac CO PCR khuan lac duang tinh chi co plasmid pUAST-dUCH gieng va tiep tuc cho san pham PCR 980 by vai cap moi dac higu cho gen dUCH (Hinh 3, gieng 2) Plasmid tai to hqp dugc cat vai EcoRI va Xhol (Hinh 4, gieng 4) cho hal bang co kich thuerc bang pUAST ma yang (Hinh 4, gieng 2) va bang co kich thuorc bang vai gen dUCH (Hinh 4, gi#1g 3) Plasmid ding cho san pham PCR vai moi dac hign dm gen mOt bang dUng kich thuoc Nhu vay gen dUCH da (long hoa ding vao plasmid pUAST Plasmid pUASTdUCH tai to hap , &rag giai trinh to kiem tra (Macrogen, Han Quoc) va vi tiem vao ruoi nham tao ruoi mang phirc hqp UAS-dUCH Bien hien dUCH gag doing ruoi chuyin gen Sau chuyen gen bang vi tiem, cac dong ruoi chuyen gen dugc tux& ch9n dtra vao gen chi thi w+ co kieu hinh matt mat dO Bon (long ruoi du dotin co mang gen mug tieu (thong qua man mat) #12-4, #234, #50-5, IA #59 dugc lam thufin va xac dinh nhiem sac the mang gen mix lieu (UAS-dUCH) Phirc hqp gen UAS-dUCH co the chuyen gen dugc khu6ch ct4i PCR vai cap moi dac higu bao quanh vitng gen UAS-dUCH ,(Hinh 5A) Theo ly thuyet, dtroi tac dOng yeu to chuyen vi gen ,P, k plasmid plIAST-dUCFI dugc dtra van phoi ruoi, vori hoat Ong cua transposase, phirc, hqp UAS-dUCH Dam girra cac trinh to IR dm y4u P dirge ellen van darn sic the ciia ruoi giant Theo thiet kg thi nglii4m chuyen gen, transposase chi hign dien cluy 409 Phan Nguyen Thiry An et al nhAt the he P (phoi ruOi) khi,chuyen gen va hohn town khong hien dien cac the he sau Do do, gen then vao nhiem sac the ton tai On clink Nhimg clang ruoi chuyen gen va ruOi hoang dai dirge lai Western de „kiem tra su bieu hien dUCH protein veil ruoi chuyen gen (Hinh 5B) Buong trimg cua cac dong ruOi chuyen gen va dong hoang dai dime thu nhan vi protein dUCH dirge bieu hien nr.inh a buong treng ruoi Khang the dUCH nhan dien dime dUCH protein (-30 kDa) a ruOi chuyen gen va mei hoang tai mil milt hitn dish CMR-CAL4 dimg ruoi Sir climg, dong rtiOi GMR-GAL4 mang promoter chuyen biet cho ino mat cho phcp xem x61 sir bieu hien vtrat mire protein mile tieu cr dia phan sinh mat ruoi, hinh thai mat e ruoi trueng thimh, ding iglu phan tich sau hen ve lac dOng cua protein mile lieu trinh te bao va sir biet Ilea cite to bao ci mire dO mo phan sinh De thu nhan cac ca the ruOi chuyen gen by mang dOng thei hai y6u to UAS-dUCH va GMR-GAL4 ca the, ma to hqp gen mang promoter GMR va GAL4 nam tren nhiem sac the X, cho nen ruoi due mang phirc hqp gen UAS-dUCH dirge lai vai mei cai GMR-GAL4 Do ca the ruoi cai mang GMRGAL4 dong lir tren nhiem sac the X nen tat ca mei a‘th'e heF I deu mang GMR-GAL4 Ban dau, mire di,,bieu hien protein dUCH ruoi chuyen gen dirge kiem tra bang lai Western giira protein dirge tach tir clic dia tien phan sinh mat ci~a au trung the ba va khang the killing dUCH, cac (long mei chuyen gen co sir bleu hien vuo mire protein dUCH 6A) •Ngoai ra,, hinh thai mat eita mei twang la kieu hinh mat nhan (rough eye) cac dong ruOi chuyen gen dUCI1 so vei kieti hinh mat hinh thtrOng ruoi holing dai Minh 613) Protein dUCII co the &rye bieu hien Niro mire, gay dl e to bat) va gay nen sir hien dill qua trinh hinh than!) mat () clic (long mei chuyen gen protein (ItICI I hien hien manh e viing phan bao the cap, not dOng vai trig (pan trong qua trinh hinh ' e ■ inig (tali (posterior) cua dia lien phan sinh mat (I link 614 by 10000 8000 10 - 8800 44 1000 980 Se 100 B Hinh San pham PCR gen dUCH (1A), pUAST/EcoRI, Xhol (3B) pUAST (2B), thang DNA churn kb (1) Hinh San pham PCR khuan lac vOi moi wizl/wiz2 100 by va khuon pUAST (ch(:mg am, 2), khu6n nine (3), khu6n cac khuan lac sang IQC (4-10), thang DNA chuan (1) 1000 by > 4110111110 Hinh San pham PCR plasmid vdi moi dac hiOu cua gen dUCH vdfi khu6n gen dUCH (2, chtitng duteng), khu6n pUAST (chi:mg am, 3), khu6n plasmid tach lir cac khuan lac (4-8), thang DNA chuan 100 by ( 41 410 Nab Hinh San pham cat bang EcoRI va Xhol cua pUAST (2), gen dUCH (3), pUAST-dUCH (4), San ph6m PCR cua pUAST-dUCH bang cap mai dac hie u gen (5), thang DNA chuan kb (1) Phan Nguitn Thuy An el al KET LUAN Gen dUCH &me tOo (long ding vao plastid pUAST va chuyen vao co the ruoi giam, tao ruoi giam chuyen gen ino hinh mang gen dUCH K'et qua butrc &Au da cho 11)4 mei lien quail giira mut dO bleu hien protein UCH-LI va kieu hinh thai milt ruoi Hon nira, me hinh chinh IA nen tang cho cac thi nghiem phan tich vai tro elm protein UCH-LI doi vai benh Parkinson ye sau Uri cam an: Di; thi dime dum hic;11 rcii sir hci Iry kink phi lir qui' Nathsted yet str.ho try gindt rriu pluMg nghiOn thud, nhii;nt sac the' Viers thmit Caw nghe Kyoto Nkil Ban TAI I.Ilal TIIAM KI IA0 Amerik AY, I lochstrasser M (2004) Mechanism and function of deuhiquitinating enzymes Rimhini Biophlw ,h o, 1695(1-3): 189-207 Corti 0, I lampe C, Darios 1,, Ibanez P, Ruberg M, 13rice A (2005) Parkinson's disease: from causes to mechanisms C R Biol 328(2): 131-142 Dawson TM, Dawson VL (2003) Molecular pathways of neurodegeneration in Parkinson's disease Science 302(5646): 819-822 Gasser T (2007) Update on the genetics of Parkinson's disease Mov Disord 22 Suppl 17: S343-350 Hegde AN, Upadhya SC (2007) The ubiquitin-proteasome pathway in health and disease of the nervous system Trends Neurosci 30( I ): 587-595 Larsen CN, Krantz BA, Wilkinson KD (1998) Substrate specificity of deubiquilinating enzymes: ubiquitin Ctermi nal hydrolases Bioehemisily 37(10): 3358-3368 Leroy E, Boyer R, Auburger G, Leube B, Ulm 6, Mezey E, Harta G, Brownstein MJ, Jonnalagada S, C'herno a T, Dehejia A, Lavedan C, Gasser 'I', Steinbach PJ, Wilkinson Polymeropoulos M11(1998) The ubiquitin path%‘:1) in Parkinson's disease Nature 395(6701): 451-452 Maraganore DM, Farrer M.I Hardy JA, Lincoln Si, Mel )(mita SK, Rocca WA (1999) Case-control study of the uhiquitin carboxy-terminal hydrolase LI gene in Parkinson's disease Nei/rot/wt 53(8): 1858- I 860 Nishiklma K I.i II, Kawamura R, Osaka II Wang YL, Ilara Y, Ilirokawa '1', Manago Y, Ammo Noda M, Aoki S, Wada K (2003) Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase 1.1 variants Biochem Riophys Res Common 304(1): 176-183 Wilkinson KD, lee KM, Deshpande S, Duerksen-I lughes P, Boss JM, Pohl J (1989) The neuron-specific protein POP 9.5 is a uhiquitin carboxyl-terminal hydrolase Science 246(4930): 670-673 ESTABLISHED THE TRANSGENIC FLY LINES CARRYING INVESTIGATE THE ROLE OF UCH-LI IN PARKINSON DISEASE UAS-DUCH Phan Nguyen Thuy Ant, Dang Thi Phuong Thaol', Masamitsu Yamaguchi2, Tran Linh Thuocl University qf Science, Vietnam National University Ho Chi Minh City 'Kyoto Institute of Technology Japan SUMMARY Parkinson's disease (PD) is a progressive neurodegenerative movement disorder that affects the population older than 65 years of age and the death of dopaminergic neurons in PD patients lead to the impairment of movement Now a day, several studies have found mutant forms of genes linked to familial forms of PD which suggests that oxidative stress, mitochondria! dysfunction, impairment of the ubiquitinproteasome system, and protein aggregation may contribute to PD pathogenesis Among them, UCH-LI is one of the most abundant proteins in the brain and plays an important role in ubiquitin-proteasome system in order to degrade the unfolded proteins in the cell Despite much effort over the last decades, the relative between UCH-LI and other neurodegenerative diseases such as PD is still unclear In other to investigate the role of' UCH-LI, we expressed dUCH, a homolog of UCH-LI in Drodophila, in transgenic flies and studied its function By constructing vector pUAST-dUCH and microinjecting into Drodophila, we created the transgenic fly lines carring UAS-dUCH and expressed dUCH in specific tissue via GAL4-UAS system Crossing UASdUCH lines with GMR-GAL4, we found that dUCH is overexpressed in posterior of eye imaginal disc, an • Author, ,fbr correspondence: E-mail: thaodp(i4hcmuns.edu.vn 412 TO Top chi Cling nghe Sinh hoc 10(3): 407-413, 2012 duch•IINICS 11 P3' ■■,1 A 1111111110Z11 NT s1 Isti Cs #12-4 #23-4 #50-5 #59 V11.1 I B III II 12 13 14 15 67 43 Om -• 30 411111Wesir Hinh A: Dien di d6 san pham PCR gen UAS—dUCH a cac clang ru6i chuyen gen sex dung khuon pUAST-dUCH (2, 3, chtirng &rang), khuon ntrac (4, 5, cherng am), khuOn be gen CantonS (6, 7, chirng nei), khuon be gen #12-4 (8, 9), khu6n be gen #23-4 (10, 11), khuon be gen #50-5 (12, 13), khu6n be gen #59 (14,15) v6i cop m8i dac hieu plasmid (gi6ng ch5n) ve m6i doc NO gen (gi6ng le tar 1) thang DNA char.' 100 by (1) B: Lai Western dUCH protein bieu hien ru6i dui* gen vai khang the khang dUCH A (-) #12-4 #23-4 #50-5 #59 +s■—•■■■••••■, Tubulin —.4 dUCH #50-5 B CantonS # 12-4 #23-4 #50-5 C Hinh Bieu hien dUCH djnh ht.r6ng tai m6 mat deng ru6i GMR-Ga14 A, Mirc de bleu hien dUCH dia tien phan sinh mat ru6i hoang dai (-) va ru6i chuyen gen dUCH B-C, Hien ttrqng mat nharn ru6i hoang dai CantonS va ru6i chuyen gen dUCH protein dUCH dtrqc Neu hien vtrqt mat ru6i (B) Hang tren va hang dtr6i twang &rig v6i de ph6ng dai 200x va 700x (C) Nht.tem dia tien phan sinh mat cue ru6i hoang dai va ru6i chuyen gen bang khang the khang dUCH.(a): dia dtrqc chip & vat kinh 10x; (a'): dia dtrqc chip vat kinh 40x Mai ten:protein dUCH dtrqc bleu hien manh second mitotic wave va du6i mat 411 Tap chi Ging nghe Sinh hoc 10(3): 407-413, 2012 important part to the eye development; therefore, the overexpression of dUCH affected the eye phenotype Moreover, transgenic flies earring UAS-dUCI1 can he a model tbr further investigation of Parkinson disease Tir klukr gene dUCH, Drosophila GA4R-GAL4, genetic tronsjOrtnotion eve specific expression 413 ... lieu (UAS- dUCH) Phirc hqp gen UAS- dUCH co the chuyen gen dugc khu6ch ct4i PCR vai cap moi dac higu bao quanh vitng gen UAS- dUCH ,(Hinh 5A) Theo ly thuyet, dtroi tac dOng yeu to chuyen vi gen ,P,... EcoRI va Xhol cua pUAST (2), gen dUCH (3), pUAST -dUCH (4), San ph6m PCR cua pUAST -dUCH bang cap mai dac hie u gen (5), thang DNA chuan kb (1) Phan Nguitn Thuy An el al KET LUAN Gen dUCH &me tOo (long... giam chuyen gen mang phirc hqp UAS- dUCH phut vu cho nghien cuu vai tre dm protein UCH- LI doi fed benh Parkinson vi xem bieu hien vtayt mirc ciia protein dUCH ruoi ruoi giam chuyen gen VAT LIEU
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