Tạo dòng ruồi giấm chuyển gen mang phức hợp UAS DUCH để nghiên cứu vai trò của protein UCH l1 đối với bệnh parkinson

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

2 Vien

TOM TAT
Benh Pakinson la benh thoai hoa he than kinh van,d6ng ph6 Wen nguiri cao tuoi, nguyen nhan la do 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 trong
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 6 nao, co vai tro quan trong trong he
th6ng ubiquitin-proteasome nham phan huy cac protein gap cuan sai trong to bao. Trong nhung nam gan day,
mac du nhieu nghien cuu da duqc thuc hien de tim ra moi quan he eirra UCH-L1 va benh Parkinson ding nhtr
cac benh thoai hoa than kinh khac, tuy nhien van de nay van con 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 ra 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 trong qua trinh hinh thanh 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 nay 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 pho 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 nay va
quan trong han nirra la cac nghien ciru nay 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 nay gift') hieu duqc mei
met so co the vii nhau va su lien he yea co the phat
sinh benh Parkinson trong to bao, nhu stir tich tu asynuclein, su thity phan chai ubiquitin trong 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 trong nao (khoang 1% - 2%) (Wilkinson et aL,
1989), thuec. ho enzyme co vai to trong viec hinh
ket i dau C dm protein trong quy trinh
thanh

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 ra khoi protein tnrec khi protein

vao he thong phan cat proteasome, ben canh do
UCH-L1 con phan tach chit& ubiquitin thanh 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 trong
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), tuy nhien van de nay van con 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 pho bien trong nghien ciru y - sinh hoc hien
nay. 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
trong 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
trong 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 0 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
trong met vector co the chuyen vi nha nhan to P, co
gen Amp` co Ichi nang Ichang Ampicillin giup sang
19c trong qui trinh tao dang.

phenol: chloroform, clio Id NIA lac rung. Sau khi 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 3 M Na acetate pH

5,2 NIA 500 ml 99% ethanol va u 30 phiit 6 -20 C, sau
do ly tam 30 phut Yeti 13000 vong/phirt 6 4°C. Tim
duct Kra lai bang 500p1 70% ethanol, ly 6'11130 phiu
vai 13000 vang/phiit 6 4°C. Tim duct de kilo to
nhien va giii trong 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 5 ill dem 10x
PCR, 5 gl 8 mM dNTP, 5µl 25mM MgCl2, 1 ul
DNA be gen (Y ng), 1 gl 1 unit Taq polymerase vi 1
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 trong 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
6 -20 C qua dem: Sau do, hoa trong 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
1 gi6 6 37 C. Sau khi 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 là ruoi dot bien mat
tang). Ru8i duqc nuoi trong mai truing de trong 2
- 3 ngiy viz& khi 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 khi tiem
phii de ruoi de trong trong 20 - 40 phut trong mai
truing thach SY. Lop ming diy bao quanh trong
du9c loai be giitp cho qui trinh vi tiem de ding.
Helper P - element vi plasmid mang nhan to P
duqc tiem vio trong 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 khi vi tiem dugc cho troy lai moi
tetrang SY de trUng Oat trien. Moi ken ruoi duce

nuoi trong mOt Ong moi trtrang, de chung na thanh
ruoi trtremg thanh hoac chi cho den khi ken no thanh
ruoi truong thanh thi bat trinh nu. Toan b0 the 114 Fo
co matt mat trong. Moi con cai Fo dugc giao phoi yen
ba con duc W 1118 va moi con due Fo dugc giao
phoi vht ba con ,cai WI 11 8. The 114 F1 c6 kieu hinh
sau: 1. Tat ca the h4 con Fi co mau mat tang. Day la
nhimg con ruoi khong dugc chuyen gen. 2. MOt so
ruoi F1 (khoing10%) mat c6 sac to do. Day la dong
ruoi dugc chuyen ,gen. 3. The 114 ,F, (han 50%) co
nhieu kieu mau mat khac nhau. Dieu nay 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 khi sang I9c de thu dugc dong ruoi chuyen gen,
cac dong ruoi nay 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 trong dung dich sira gay 5% trong PBST
(80 mM Na,HPO4, 25 mM Nal-14304, 100 mM NaCI,
0,1% Tween 20), lac nhe trong I gia cho dung dich
ti4p Vic deu vori be mat dm mang lai. Sau do, ngain
mang lai trong khang the khang dUCH (1/10000)
trong 1 gi6,dong thai lac nhg nhang hop. Sau do rira
ki mang 5 Ian trong dung dich PBST, moi Ian 5 phin.
Tiep tuc u mang vai khang the tit& cap danh dau
bang HRP pha loang trong dung dich PBST vai ti 14
1/20000 trong I gia, dong thai lac nhe hop. Sau do

rim ki mang bang dung dich PBST 5 Ian, mai lin 5
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 3
trong PBS (140 IAM NaCI, 3 ttM KCI, 8 ttM
Na2HPO4, va 15 pM KH,PO4 , pH 7,4) wen da. CO
dinh dia trong 2% paraformadehydc trong PBS 6
4 C qua dem., Sau d6 rira 6 Ian bang PBS 0 nhigt
de) phong. U vori khang the sa cap a-dUCH
antibody (1/10000) 0 4 C qua dem. Rita 6 Ian bang
PBS va 0 yeti khang the (ha cap a-FITC antibody
(1/1500) 4 gio. o ‘nhigt dO phong lioac .4 C qua
dem. Rim 6 Ian bang PBS va ii mau trong 100%
glycerol trong 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 ra plasmid tai to hqp pUASTdUCH. Plasmid dugc !Dien nap E. coil DI-15a tao ra
cac (long E. coil DH5a/pUAST-dUCH.
Kiem tra khufin lac bang PCR vai cap mo
ll
wiz] /wiz2, cac khuin lac 6 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 5 khuan lac CO PCR khuan lac duang
tinh chi co 2 plasmid pUAST-dUCH 6 gieng 5 va 6
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 nay ding cho
san pham PCR vai moi dac hign dm gen mOt bang
dUng kich thuoc. Nhu vay gen dUCH da (long hoa
thanh 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 khi 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 trong 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, khi
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 trong the. he P (phoi ruOi) khi,chuyen gen va
hohn town khong hien dien cac the he sau do. 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

1

2

mang

dOng thei hai y6u to UAS-dUCH va GMR-GAL4
trong 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 con 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 ra 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 thanh la kieu hinh mat nhan (rough
eye) 0 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 trong 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 trong qua trinh hinh thanh
' e ■ inig (tali (posterior) cua dia lien phan sinh
mat (I link 614

1 2 3

by
10000
8000

8 9

2 3 4 5 6 7

10

- 8800

44

1000

980

Se

100

B
Hinh 1. San pham PCR gen dUCH.

(1A),
pUAST/EcoRI, Xhol (3B). pUAST (2B), thang DNA
churn 1 kb (1).
2

3

4

5 6

7 8

Hinh 2. 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).
2

3

4

5

1000 by >

4110111110

Hinh 3. 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 4. 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 1 kb (1)


Phan Nguitn Thuy An el al.
KET LUAN
Gen dUCH
&me tOo (long thanh 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 nay 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.
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(2005) Parkinson's disease: from causes to mechanisms. C
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Dawson TM, Dawson VL (2003) Molecular pathways of
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Gasser T (2007) Update on the genetics of Parkinson's
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Larsen CN, Krantz BA, Wilkinson KD (1998) Substrate
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Leroy E, Boyer R, Auburger G, Leube B, Ulm 6, Mezey
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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 2 1 4 5

1.

B
8

III II 12 13 14 15

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30

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Hinh 5. 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 6 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 6. Bieu hien dUCH djnh ht.r6ng tai m6 mat 6 deng ru6i GMR-Ga14. A, Mirc de bleu hien dUCH 6 dia tien phan sinh mat
ru6i hoang dai (-) va ru6i chuyen gen dUCH. B-C, Hien ttrqng mat nharn 6 ru6i hoang dai CantonS va ru6i chuyen gen
dUCH khi protein dUCH dtrqc Neu hien vtrqt min 6 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 6 vat kinh 40x. Mai ten:protein dUCH dtrqc bleu hien manh 6 second mitotic
wave va 6 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

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