Tạo mô hình ruồi giấm chuyển gen biểu hiện protein α synuclein nhằm ứng dụng trong nghiên cứu cơ chế phát sinh bệnh parkinson

Tgp chi C6ng nghe Sinh hoc 11(3): 425-432, 2013

TAO MO HINH RUOI GIAM CHUYEN GEN BIEU HIEN PROTEIN a-SYNUCLEIN
NHAM tiNG DUNG TRONG NGHIEN CtrU CO CHE PHAT SINH BENH PARKINSON
Nguyen Thi Ttrimg Vy, flung Thi Phircmg Than, Trim Linh Think
Triremg Doi hoc Khoa hoc to nhien, Dgi hoc Quac gia Thanh ph a Ho Chi Minh

TOM TAT
Bing nhieu mo hinh nghien ciru khac nhau, dac bi0t la me hinh d6ng vat chuyen gen, cac nha khoa hoc
dang ngay tang co nhieu brat tien trong nghien ciru b0nh di truyen a ngtrai va co the sinh hoc phin tir cua
bOnh. Vai nhieu tru diem not trOi, mo hinh ruoi giam Drosophila melanogaster da va dangduqc sir dung nhieu
trong nghien ciru y sinh. Nhieu ke't qua nghien ciru benh thoai hoa than kinh, ung thu, tieu &rang, tim mach,
tren mo hinh ruoi giant da duqc cong bo ten cac tap chi khoa hoc nhu Nature,
roi loan bien
Science..., Clang gap lan cho cac hieu biet khoa hoc ye ca the b0nh. Mic du \fay, tai V* Nam, viec tat) va sir
dung cac mo hinh mai giam chuyen gen con kha mai, hiu nhu chua cOsphOng thi nghiem nao trien khai. Trong
bai bao nay, chung trinh bay cac ket qua nghien ciru too mo hinh ruoi giim chuyen gen a-synuclein (SNCA)
nham img dung trong nghien turn ca the benh Parkinson. Cac cong b8 tnrac day cho thiy hi0n din cita the
viii Lewy, c6 thanh phan chit yen la protein a-synuclein, lam thoai h6a din cac to bao thin kink san sinh
dopamine va IA nguyen nhin chit yeu gay ra 130nh. Bang cac ky thuit sinh hoc phin tir thing toi chuyen cDNA
gen a-synuclein cua ngtrai vao trang ruoi giim da thy tinh nham sang loc thu nhin cite dang ruoi mang gen
SNCA. Ch'ing toi di to thanh ding ba dung ruoi mang gen SNCA va hat dang d6t bien thay the acid amin
A3OP va A53T. Han the nua, chung toi cOngda thanh cong trong viec sir dung he thong UAS-GAL4 nham
bleu hi0n.dinh htrang gen SNCA nao ruoi nham tim hieu ca cite tic dOng cita str Lich to SNCA tren hoot dOng
dm 110 than kinh trong cac nghien ciru sau.
Tir Wet: Benh Parkinson, a -synuclein, the Lewy, Drosophila melanogaster, he thijng UAS-GAL4

DAT VAN DE
Benh Parkinson (PD, Parkinson Disease) la benh
thoai h6a thin kinh gay ra bii sv thieu hitt dopamine
trong nao gitza. Tuy tinh hinh benh hien nay rat pito

hien, Ian reng va mang tinh nghiem trong, nhung
nhfing nghien ciru ve nguyen nhin giy benh, lieu
phap phong benh, cac mo hinh nghien ciru mo ph6ng
va trj benh van chua mang Igi nhang dap an mong
dqi. Nhieu ciu h6i Ian ve benh Parkinson van chua
co ciu tra lai. Nhang nghien ciru gin day bat diu
hiring ve mgc tieu phatshien nhang det bien don co
lien quan den sv di truyen cga met so kieu benh PD,
dieu nay cho phep chgng ta hieu sau han ve ca the
phan tir cua benh PD thong qua viec thiet lip cac mo
hinh nghien ciru sir dung deng vat chuyen gen co
kieu hinh benh PD (Feany, Bender, 2000; Bilen,
Bonini, 2005; Pendleton et al., 2002). Khong nhang
co the dap ung nhu cau nghien ciru cac nguyen lY
giy benh a mac sinh hoc phan tir, mo hinh nghien
dm nay con cho phep nghien ciru sang loc cac chit
hem hoc co host tinh tri benh (Sang, Jackson, 2005).
MO hinh chuet chuyen gen da va dang duqc sir
dung Ong rai de nghien ciru cac benh tren
Tuy nhien, mo hinh nay thuing ton nhieu chi phi

cling nhu can met khoang thin gian kha dal. Met
trong cac &di tuang sinh vat co the sir dung lam m6
hinh nghien ciru berth tren ngtrii la mo hinh ruoi
giam Drosophila chuyen gen. Day la met mo hinh
huu ich cho viec sang loc rung rat cac gen giy benh
vii chi phi thip va thii gian ngan. 0 mo hinh ruoi
giim chuyen gen chung ta co the nghien ciru va mo
piling met cfich ro rang mang Itrai cac gen lien quan,
dieu Ma gen giy benh. Vai cac tru diem nay, mo

hinh ruoi giim chuyen gen da va dang duqc irng
dung trong cac nghien ciru benh tren ngtrii (Bilen,
Bonini, 2005; Pendleton et al., 2002; Sang, Jackson,
2005).
Benh Parkinson thuing duqc biet den IA benh
liet rung, day la benh thoai hoi than kinh theo deo
tuoi, gay anh hiring den kha nang van &ling, giti
thang bang va kiem sok ca the dia benh nhan. 6
viot Nam, day la met benh tuang del ph6 bien vii tSt
10 mac Will la 90-100 ngtrii ten 100000 din. Sr
thoai hela dan cac to bao san sinh dopamine duqc cho
la nguyen nhin chinh don den benh Parkinson
(Gandhi, Wood, 2005; Moore et al., 2005). Yeu to
moi truing va yeu to di tuyen co lien quan chat the
tai sir thoai hia nay.
425


Nguyen Thi Ttrong Vy et al.
Cie nghien cuu cho thiy sir hinh thinh the WU
Lewy IA nguyEn nhan chinh dAn dEn cic r8i loan
chirc nang ty the dua den stress oxy h6a cOng vai
viEc dopamine bi oxy h6a vi tra nen gay d'QC cho to
bio (Feany, Bender, 2000). Gia thiet dtra ra li asynuclein, parkin vi DJ-1 c6 lien quan den qua trinh
tich to oh protein de hinh thinh nen the Lewy.
Trong do a-synuclein &rig vai tro trung tam trong
bEnh Parkinson vi day la thinh phan chinh trong the
Lewy (Rosner et al., 2008).
Protein synuclein dugc biet den vi cong b6 nim
1988 bed Maroteaux khi nghien cuu ve loii Torpedo

californica, co cic clang chinh IA a-synuclein, (3synuclein, vi y- synuclein, synoretin; dugc tao ra do
qui trinh splicing de hinh thinh mRNA tnrang
thinh. Trong do a-synuclein co vai tro khi quan
trong trong qui trinh diet' haa chu trinh hinh thanh
tni tiet tai synap vi dieu hoa tier dopamine (Perez et
al., 2002). Cac nghien et:1.0 in vitro cho thay clang
monomer khong gap cuOn c6 the tich to thinh cic
Bang oligomer nh6, cau tnic nay co the duce on dinh
bai nhang twang tic giong u-sheet vi sau d6 hinh
thinh clang co trong lucmg phan tir cao han, khong
tan goi la fibril, day IA clang ,cau trite dugc tim thay
khi pito bien trong cic the Lewy. Ngaii ra cic
nghien cuu con cho thay khong chi so tich to cila
protein a-synuclein dugc tim they trong cfic the
Lewy dic trung cho bEnh Parkinson ma can co cic
dot bien khfic ten gen ‘SNCA
h6a cho asynuclein. Cac dang
bien nhu sau: dang dOt bien
diem amino acid alanine bi thay the bai threonine a
vi tri protein thir 53 (A53T)hoic proline tai vi tri
protein thu 30 (MOP), mOt so it trtrang hqp là dang
amino acid glutamic bi thay the INA lysine tai vi tri
protein thir 46 (G46L), vi dang dOt bien lim gia tang
so lacing bin sao cOa gen SNCA clan tai du thira
Itrang a-synuclein (Polymeropoulos et al., 1997).
Tuy vEy, ca the dm so tich to a-synuclein trong
mOt vai, tnrang hgp
bEnh Parkinson van china
dugc hieu mOt cach day du. Do de, viEc xay dung
m8 hinh nghien dui so tic eking cua gen a-synuclein

len hE than kinh co vai fro then chat trong viEc tim ra
ca the phan tir phit sinh bEnh Parkinson.
CS nghien cuu nay, chimg tai sir dung ruoi giim
de xay dung m8 hinh nghien cuu bEnh Parkinson.
Trong nhang nim qua, nh6m cic nhi khoa hoc Chau
Au vi M9 di xay dirng m8 hinh ruoi de.nghien tiro
bEnh Parkinson nhu m8 hinh ruoi chuyen gen bieu
hiEn vuot mire gen a-synuclein, DJ1, parkin...
(Feany, Bender, 2000). Mic du k9 thuit, mo hinh
ruoi gam chuyen gene a-synuclein mang tinh ke
426

thira cic thinh tkIll di cong b6, nhung tai ViEt Nam
day li nghien ciru dau tien htrang tai viEc sir dung
m8 hinh ruoi giam de nghien cuu bEnh Parkinson.
Ket qui nghien cuu cling là tier' de cho viEc thiet ke
cic mo hinh ruoi giam chuyen gen de sang icc cfic
chat chiet xuat to nhien dE tri bEnh Parkinson tai
phang thi nghiEm cua cluing VOL
VAT LIEU VA PHYONG PHAP
\It liEu
Vector tai to hop pBlue/SNCA (ViEn Khoa hoc
Cong nghe Kyoto, Nhit Bin) mang cDNA dm gen
SNCA ma h6a cho protein a-synuclein.
Vector pUAST (ViEn Khoa hoc C8ng nghE
Kyoto, Nhit Ban) mang gen kiting khing sinh amp'
giup cho viEc sang 19c cic the bien nap mang vector
tai to hg,. Wing MCS, viing,chira duy nhat mOt vi tri
nhin biet
cic enzyme cat giai han can thiet cho

Bong h6a gen mac tieu vio vector. Ngoii ra, ten
vector can c6 cic yEu to cho phep then gen vio
nhiem sic the ruoi giant nhu: nhan to chuyen gen P,
gen chi thi w cho phep khoi phut tinh trang mat de a
ruoi giam dOt bien gen mit trang, trinh to UAS
(Upstream Activation Sequence).
Chung Escherichia coli DH5a [FendA1 hsdR17
(rklmk") supE44 thik-recAl gyrA96 A/acUl 69 (T80
lacZ AM15)] (BO mon C8ng nghe sinh h9c phan tir
va mai truang, Twang Dai hoc Khoa hoc To nhien,
Dai hoc Quoc Gia Thinh pito H8 Chi Minh) dung
trong clang h6a gen.
Cfic.doni ruoi giant Drosophila melanogaster:
dung mei giam dung trong chuyen gen A2-3, mat
tang; dang ruoi mang balancer P ( w; sp/cyoGFP;
pre/TM6B); dang P{+; Elav-GAL4/cyo; +) (8765)
bieu hiEn tai mo nio c6 kieu hinh canh cong; clang
P{+; +; Ddc-GAL4} (7009) chira promoter cfia gen
DOPA decarboxylase ginp bleu hiEn tai cic to bio
than kinh sin sinh dopamine; dung ruoi hoang dai
dai chUng CantonS
+; +} vi dOng bieu hiEn 11galactosidase P{+;+;UAS-LacZ} (Ngfin hang rail
giam Bloomington, my).
Pinning pilaf)
Tgo (long E. colt DH5a mang vector tdi to hgp
pUASTISNCA
cDNA SNCA thu dugc tir vector pBlue/SNCA
bing phin Ung PCR vai enzyme Pfu polymerase
(Fermentas, Singapore) dirge cat bang Xhol va BglIl

Tgp chi Cling nghe Sinh hgc 11(3): 425-432, 2013
(Fermentas, Singapore) va not vao vector pUAST de
tao vector tai to, hqp pUAST/SNCA. Vector tai to
hqp nay duqc ban nap vao E. coli DH5a bang Ma
bien nap va sang 19c tren mai trutmg LB-Amp100.
Khuins lac m9c tren moi truimg LB-Amp dtrqc
sang 19c bang phuang phap PCR khuin lac vai cap
mei teen plasmid bao quanh vi tri ellen gen
wizl/wiz2. Sau do, nheing khuAn lac cho san pham
PCR co kich thuac twang img vai kich thu6c.dq
thu nhan plasmid bang
doin se duce nuoi cay
phucmg phap SDS-kiem. Vector thu duac tit cac
khuan lac tren duqc PCR viii cap mai dac hieu gen
va kiem tra bang enzyme cat giai han XhoI va Bg111.
Trinh to gen muc tieu tren vector tai to hop duqc giai
trinh to bang phucmg phi') Sanger va phan tich bang
phan mem Jellyfish.
Phwong phdp Igo m6 hinh ruoi gam chuyin gen
Vi tiem
Thu trimg ru6i cua clang ruoi A2-3 mat trang
mai duqc thy tinh 6 giai doan 0-3 gibs va dux hien vi
tiem vector tai to hqp pUAST/SNCA vat) phan duoi
cua trimg b nhiet de 18°C. Trimg mai sau khi vi tiem
duce nuoi tren moi truang dinh threw 6 25°C thu ca
the Fo.
Sang 19c ruoi mang vector tai to hQp cla sat nhap vao
b0 gen
Ruei Fo duqc thu nh'gn va lai vbi ruoi det ban

mat trang W118 nham Oat hien the he ruoi lai F 1
mang gen awe tieu co kieu hinh mat do do gen chi
thi w+qui dinh..Cfic clang ruoi chuyen gen sau do
ductc lai vai ruoi mang balancer de xfic dinh vi stri
clan gen tren nhiem sac the ruoi va lam thufin nham
thu cac clang citing hqp tir gen muc tieu SNCA. Sq
hien dien oh gen SNCA trong ruoi chuyen gen &lac
kiem tra bang, phucmg phap PCR yeti moi dac hieu
gen va mil bat tren nhiem sac the, bao quanh gen
muc tieu SNCA.
Kiem tra sr, bieu hiin act protein a-synuclein cua
Mc &mg mai chuyin gen tgi ma nao
Cfic clang ruoi giAm chuyin gen a-synuclein da
duce tao va kiem tra duac tien hanh thu cac ruoi
cfii con trinh va lai vai clang bieu hien protein GAL4
(sir dung clang Elav-GAL4/Cyo) nham kiem tra sq
bieu hien dm protein a-synuclein tai m8 nao. ElavGAL4/Cyo la clang ruoi mang promoter chuyen bat
cho mo nao, do do khi lai dung rail nay vai cac
deng UAS-SNCA thi chi nhitng to bao than kinh mai
bieu hien protein a-synuclein. The he con lai Fl

duqc chQn loc dip vao kieu hinh do cac balancer quy
dinh, vi vgy cac ca the mei lai Fl mane UAS-SNCA
va Elav-GAL4 duqc hp chQn co kieu hinh canh
thang. Protein tong tir cac mau dau ruoi duce thu
nhan va tien hanh dien di phan tich bang SDS-PAGE
va kiem chirng bang Western blot vsfti khang the dac
hieu khfing a-synuclein.
Deng ruoi Ddc-GAL4 kitting mang balancer va
la clang mang promoter chuyen bat gifip bieu hien

tai cac to bac, than kinh san sinh dopamine. Cfic the
he ruoi lai F 1 dime sir dung khao sat str bieu hien
protein a-synuclein tai cac m8 than kinh chuyen biet
san sinh dopamine bang phuang pita!) hem m8 man
dich huStnh quang vai khang the dac hieu khfing asynclein.
hoa m6 mien dich huynh quang
Nhu
(1 nun unoh istochemistry)
Ruoi lai Fl duqc 25 ngay tuei duce chQn dimg
de Lich nao va nhuOm hoa m8 mien dich huStnh
quang. Nao ruoi duqc tech trong dung dich PBS va
co dinh mo bang dung dich paraformaldehyde 4%
pha trong PBS 6 nhiet dO phong 30 phut. Sau (16 rira
bang dung (Lich PBS-0.3% TritonX100 (v/v) va khoa
mitt mo bang dung dich huyet thanh de (Normal
goat serum) 10% trong PBS-0.15% TritonX100 (v/v)
nhiet de phong 30 phut. MAu m8 nao duqc it yeti
kiting the so cap khfing a-synuclein b 4°C qua dem.
Sau do matt m8 duqc rira 4-5 lan bang dung dich
PBS-0.3% TritonX100 va duqc u vai khang the thir
cap phat huknh quang FITC 484nm b nhiet de ?hong
2-3 gi0. Mau nao duce rira sach va Lich nao co dinh
len lame va quan sat clued kinh hien vi hujrnh quang.
Hinh inh &Km phan tich bang chtrcmg trinh ImageJ,
thong ke bang kiem dinh t-test cita Microsoft Excel.
KET QUA VA THAO LOAN
That ke vector mang va Men MO gene SNCA
trong ruoi gigm
Chung Vol tan hanh thu nhgn cDNA gen SNCA
bang phan Ong PCR tir khu8n mau là plasmid

pBlue/SNCA va clan vao vector pUAST bang phin
Ong not nha enzyme T4 DNA ligase. Vector .tai to
hqp pUAST/SNCA sau do duac sang loc va kiem tra
bang phuang phfip PCR vai mei (lac hieu va phuang
phap cat giai han. Ket qui kiem tra vector tai to hqp
cho they vector nay mang gen SNCA the hien tren
san pham PCR kich thuac 435bp ttrcmg Ong vai kich
thuac cDNA gen muc tieu (dit lieu thqc nghiem
427


Nguyen Thi Tuang Vy et al.
kitting trinh bay tai day). D6ng thtri ket qui xir ljr yeti
enzyme cat ihi han va ket qui kiem tra trinh tir gen
ding cho thay gen SNCA duqc chen vio vector co
trinh ttr giOng 100% vei trinh ti,r cDNA cua SNCA
ten ngan hang gen. Nhu vay, chang toi da tao clang
thanh tong vector tai to hqp mang gen SNCA. Ben
canh do, cic ket qui &mg hoi wan tu tren hai (long
gen SNCA det bien ding cho thay chung Col da thinh
ding trong viec thu nifin hai vector mang 2 clang On
bien A3OP_SNCA vi A53T SNCA. Cfic vector nay
sau d6 dtrqc tich chiet tit ti bao E. coil di vi tiem
vio trong ru6i.
Sang loc tool mang vector tai
vio bQ gen

a hop (la sat nhap

Sau khi vi tiem, nhO vio yeti to chuyen vi

transposon P tren vector pUAST (Bachmann,
Knust, 2008), gen SNCA duqc sat nhip vector, tai
to hqp met cfich ndu nhien vio be gen ruoi giam.
Cic &mg ruoi chuyen gen c6 kieu hinh mat do
duqc lua ch9n clua vio gen chi thi
Chang UM
tien hinh lam thufin cic clang ruoi chuyens gen thu
nhan duqc vi sir ding phep lai voi ruoi mang
balancer de, xic dinh vi tri gen chen tren nhiem sac
the rail giam, ket qui phan tich cho thay cluing
toi thu nhan dtrqc 03 (long ruoi chuyen gen nhu
trinh bay tren bang 1.
Sau khi lim thuAn va xic, dinh nhiem sic the
mang gen mac tieu, chfing toi tiep tic tien hinh kiem
tra phan tich cic dOng ruoi nay. Tnrac tien, chang toi
den hinh kiem tra su hien dien cita phirc hqp gen
UAS—SNCA (hoac
UAS-A3OPSNCA; UASA53T SNCA) trong ca the ruoi chuyen gen bing
phucmg phip PCR vai cap moi dac hieu bao quanh
yang gen mac Lieu (Hinh 1A). Theo ly thuyet, dm%

tic Ong cua y'eu to chuyen vi gen P, khi vector
pUAST/SNCA &qv dua vio phoi ruoi, vai boat
dOng ciut transposase, phirc hqp UAS-SNCA nem
giera cic,trinhtv IR cita yeu to P se duvc chen vac)
nhiem sac the, ctia ruoi giam. Trong thiet ke thi
nghiem chuyen gen dia chimg toi, enzyme
transposase chi hien dien duy nhAt trong the he P
(phoi ruoi) khi chuyen gen. Cic the he sau do,
enzyme transposase hoin toin, kh8ng hien dien, do

vay, sir chen gen vac) nhiem sac the la su chen gen
On dinh. Chang Col ,thu nhan DNA be gen dm rail
chuyen gen du tuyen vi sir dang lim khuon trong
phan irng PCR kiem tra yeti moi dac hieu cho phirc
hqp gen mac tieu. Ket qui phin.ting PCR vai khuon
li DNA be gen dm ru6i chuyen gen cho vach co
kich thudfc nhu du doin img vai 2 cap moi khi so
sinh yeti vach chimg dtrcmg (Hinh 1B, gieng 2, 3)
nhu sau: 1 vach c6 kich thtrac 435bp img vai cap
moi dac hieu cho gen SNCA (Hinh 1B, gieng 9, 11,
13), 1 vach co kich thy& 585bp img v1i cap mei dac
hieu bao quanh viing ngoii gen wizl/wiz2 (Hinh 1B,
gieng 8, 10, 12). DOng thidong ruoi doi chtng
CantonS kh8ng c6 xuat hien vach gen ten hinh khi
PCR vai ca 2 cap moi ten (Hinh 1B, gieng 6, 7).
Mar vay, chang toi da tao (long thinh Ong 3 dong
ruoi giam chuyen gen mang gen a-synuclein vai vi
tri chen gen tren nhiem sac the nhu sau: UASWT_SNCA (nhiem sic the II); UAS-A3OP_SNCA
(nhiem sic the II); UAS-A53T_SNCA (nhiem sic
the X) (Bing 1).
•
Cic.clong ruoi nay tie!) tic duqc kiem tra khi
tang bieu hien protein a-synuclein trong mo nao
bang phuong phip Western blot vi nhuOm mien dich
huSmh quang vai khing the doc hieu khing asynuclein.

Bang 1. Cac clang ru6i chuy4n gen bi6u hiOn gen a-synuclein
STT

Ten clang ru6i chuyen gen

Phirc him gen

1

Dro-AlphaSyn WT

UAS-SNCA

2

Dro-Syn A3OP

UAS-A3OP_SNCA

3

Dro-Syn A53T

UAS-A53T_SNCA

Bien hiOn protein a-synuclein trong m0 nao
De nghien ciru chirc nang cua a-synuclein ten
mo hinh ruoi giAm chuyen gen, cluing toi sir dung he
thong UAS-GAL4 nham bieu hien vuqt mirc protein
a-synuclein tai vi tri cic mo can khio sat. He thong
nay Om 2 phAn, UAS (upstream activation
428

NST nhen gen chen

Dec diem
Deng hap
Deng hap

X

Deng hap

sequence) là loan trinh tv duvc thiet ke tnrot gen
mac Lieu, vi GAL4 dtrqc thiet ke nim sau promoter
bieu hien dinh huong mo. Benh Parkinson nhu dtrqc
biet la benh thoii hem thin kinh, do d6 chang toi
ch9n dong ruoi Elav-GAL4 giap bleu hien protein
chuyen biet tai m8 nao. Khi lai cic (long ruoi UASSNCA yeti clang Elav-GAL4, b the he F1, GAL4


-

Tap chi Cong nghe Sinh hoc 11(3): 425 432, 2013
dugc tong hcrp se gin len trinh to UAS IA gen mix
tieu dual su kiem soat cua UAS se dugc phien ma
giap bleu hien dinh huang protein a-synuclein tai m6
nao ruoi (Hinh 2A). D'ong thei dua vao kieu hinh cua
F1, chang toi chqn con lai có kieu hinh canh thing se
mang phirc hqp caa UAS-SNCA va Elav-GAL4.
Ket qua dien di SDS-PAGE mau dau ruoi truans
thanh (Hinh 2B) cho they con lai Fl,tir dong ruoi d8i
dugc chuyen cDNA SNCA
chirng CantonS

(Hinh 2B, gieng 2) kh6ng the bieu hien protein
tuang ang. ci cac meu protein ter cac con lai Fl
(Hinh 2B, gieng 3, 4, 5) dm cac dong ruoi chuyen
gen muc tieu cho they có bieu hien protein muc tieu
co kich thuac khofing 18kDa so vai thang protein
chuan.
D6ng thei ket qua Western blot vai khang the
dac hieu khang a-synuclein Minh 2B, gieng 3, 4, 5)
cho they cac d6ng ruoi lai nay deu bieu hien protein
a-synuclein. Nhu Vey, protein a-synuclein 6 clang
hoang dui va 2 dung det hien A30P, A53T deu dugc
bieu hien dinh huang thanh ding tai m6 nao cua ce
the mei lai Fl.
Mat Ichic, chUng toi danh gia su bleu hien asynuclein va cac dung det hien tren m6 nao ruoi tai
cac to bio than kinh san sinh dopamine, dua tren ca
the tong hqp dopamine (Yu et al., 2005), chimg toi

bieu hien dinh huang a-synuclein trong, to bao than
dieu hoa cua
kinh san sinh dopamine duoi
promoter Ddc (DOPA decarboxylase), met promoter
dieu hoa tong hcrp DOPA decarboxylase - enzyme co
vai tre trong ,con dueng sinh tong hqp dopamine.
Chung toi thiet lap cac dong mei chuyen gen mang
phac hqp *en Ddc-GAL4>UAS-SNCA va thu nhan
mo nao ruoi chuy'en gen, nhuem boa mo mien dich
hujrnh quang vai khang the khang a-synuclein. Ket
qua kiem tra bieu hien protein a-synuclein trong to
bac) than kinh san sinh dopamine (Hinh 3. A) cho they
met so vang tren m8 nao a ruoi mang gen bieu hien asynuclein clang khong det bien va dung det bien

A30P, A53T cho tin bleu huSfnh quango cac cum to
bao, sinh dopamine (yang dugc khoanh trim) khi doi
chieu tren m6 hinh cac yang san sinh dopamine dugc
ding be beri White ye. (long tic gia nem 2010 (Hinh 3.
A3). Cac dong mei d8i chimg Ddc-GAL4 va DdcGAL4>UAS-LacZ khong cho tin hieu lai vai khang
the khang ha-synuclein (Hinh 3. Al, A2).
Ket qua nhu'em ma dugc xac dinh ctrimg de tin
hieu bang chucmg trinh Image) va th8ng ke the hien
qua bieu d8 hinh 3. B cho they mew de tin hieu
huynh quang thu dugc ter cac (long ruoi co bleu hien
protein a-synuclein cao hen nhieu 6? 4-5 Ian so vai
cac dong doi chfrng va coy nghia thong ke khi phan
tich bang kiem dinh t-test vai cac gia tri p<0.05, n=3.

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Hinh 1. Chen gen a-synuclein vao nhiern sec th6 ru6i giAm va sang IQC (Jong ru6i chuyen gene. A: M6 phong vi tri gene
chen tren nhiern sec the ruoi gii#m ye vi tri cac cap mai dung trong sang loc ruoi chuyen gene. B: Kitt qua PCR kiem tra skr
hien then c6a gene a-synuclein trong nhiern sac the cac &mg ruoi chi* gene. Cac gieng 3, 5, 7, 9, 11, 13: Ket qua PCR
vai mai dec hieu gene a-synuclein; Cac gieng 2, 4, 6, 8, 10, 12: Ket qua PCR vbi cep mai ten nhiern sac the cua ruoi
bao quanh vi tri chart gene.

429


Nguyen Thi Tuerng-Vy et al.
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Hinh 2. Neu hian dinh huerng protein a-synuclein tren &prig ruoi giam Elav-GAL4/Cyo tai ma nao. A Bieu hien dinh hirifyng
gen SNCA tai and nao ruoi bang ha th6ng UAS-GAL4. B. Dien di SDS-PAGE va Western blot veri khang the khang asynuclein; 1. Thang protein; 2. D61 cht:rng F1{+; Elav-GAL4/+;+}; 3. F1{+;UAS-WT_SNCA/Elav-GAL4;+}; 4. F1{+;UASA3OP_SNCA/Elav-GAL4;+}; 5. F1{UAS-A53T_SNCA/+;Elav-GAL4/+;+}.

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Hinh 3. Bieu hien dinh hutfrig a-synuclein tren m6 nao ruoi tai cac vOng to bao then kinh dopamine . A. Nhu6m h6a m6
mien dich NS/F.1h quang m6 nao vbi khang the khang a-synuclein; B. Bieu d6 cirong dO tin hieu a-synuclein/pixel vbi
*,p<0.05, n=3.

KET LUAN
Cac kEt qua trinh bay tren cho thay chung toi
da thanh Ong trong viec tao dung cac vector tai to
hap mang gen ma hoa protein a-synuclein
.
pUAST-SNCA va flan hanh vi tiem thanhcong
vao
tao mo
ruoi giim Drosophila melanogaster
hinh ruoi giim chuyen gen SNCA irng dung trong
nghien ciru ca chE Oat sinh benh Parkinson. Dong
theri chUng toi Ong da thanh tong buck diu trong
viec bieu.hien protein a-synuclein tai mo nao ruoi
nham tiep can gin han vai mo hinh ruoi
430

Parkinson. Mac da mo hinh nay da dtrac Feany va
dung tic gia cong b8 tai top chi Nature nam 2000
nhang day la mo hinh ruoi giam chuyen gen dau

tien duce tao thanh ding tai Viet Nam. dieu nay
minh chirn.g cho viec
to c6 the lam chu cac
kjtsthuat de tao thanh cac m6 hinh ruoi chuyen gen
nham phuc vu cac nghien ciru benh tren ngtrati.
COng voti viec tao thanh m8 hinh nay, chimg tot se
tiep tuc nghien ciru nham lam sang to cac eau hoi
chung
gieri nghien ciru ve vai tro chirc nang
cling nhu ca the hoat Ong ctia a-synuclein trong
hoat dOng cila he than kinh.


Tap chi Gang nghe Sinh hoc 11(3): 425-432, 2013
Lei cam an: Nghien ctiu nay &ivy thus hien vol

kinh phi to DH Quik gia Tp. H4 Chi Minh. Cluing
ho try nhiet tinh ctia Gido str
tOi trdn trpng
Yamaguchi Masamitsu, Vien khoa hoc ky thuat
Kyoto, Nhat Ban.
TAI

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431


Nguyen Thi Tu6ng Vy et aL

ESTABLISHMENT OF a-SYNUCLEIN EXPRESSING TRANSGENIC FLY MODEL FOR
STUDY ON PARKINSON DISEASE MECHANISM
Nguyen Thi Tuong Vy, Dang Thi Phuong Thao*, Tran Linh Thuoc
University of Science, Vietnam National University Ho Chi Minh City
SUMMARY
By many different tools and research models, particularly transgenic animal model plays a very important
role; scientists over the world have been successful in uncovering deeply either in study on human genetic
diseases or investigation of disease mechanism. Bearing many good points for being a research model,
Drosophila melanogaster has been widely used in health science study. Numerous studies on
neurodegenerative diseases, cancer, diabetes, cardiovascular, metabolic diseases, and so on had been carried on

Drosophila model and the results of those studies had been published on Nature, Science, and so on. Those
publications gave great contribution on knowledge to understand wisely disease mechanism. Although the
transgenic Drosophila model has been widely used on the world, it is quite new in Vietnam and so far in our
country, there is no laboratory working on this model for studying human disease's mechanism. In this study,
we present our results on successful establishment of Drosophila model bearing a-synuclein (SNCA) gene for
study mechanism of Parkinson Disease (PD). It is well known that an existence of Lewy body which is created
mostly either by overexpression or mis-folding of a-synuclein protein in patient s brain caused neuron cell
degradation and stayed as main reason that causes PD. Using molecular biology techniques, we transferred
human SNCA cDNA into fertilized fly embryos in order to screen for SNCA transgenic fly lines. We succeed
on establishment of three fly lines, which carry SNCA gene and two mutant types for amino acid shifted MOP
and A53T. Furthermore, we also succeed on using UAS-Ga14 system for directly expression of SNCA gene in
Drosophila brain. The models will be useful in further study on effect of a-synuclein accumulation in brain.
Keywords: Parkinson disease, a-synuclein, Lewy body, Drosophila melanogaster, UAS-GAL4 system

* Author for corresspondence: Tel: +84 -8-38307079;E-mail:
4E32