1

Cc enzyme su dng trong

k thut to dng
1.Restriction endonuclease
2.Cac en:vme bin doi DNA
Nuclease.
ExonucleaseIII,DNaseI,nucleaseS1
DNA polvmerase.
DNA pol.I,Rtase,RNA pol.
En:vmes bin doi dau cuoi DNA.
Polvnucleotide kinase,terminal trasferase
3.DNA ligase
I. Cc enzyme ct gii han (RE)
1. Ngun gc
2. Phn Ioai
II. K thut di truyn
1. Vector tao dng
2. T bo ch
3. K thut tao dng
A. Chin Iuc tao dng
B. PCR
4. Thu vin gen
A. Thu vin b gen
B. Thu vin cDNA
Restriction Enzyme (RE)
uc tm thy trong cc Ioi vi khun
khc nhau I cc endonucIease c kh
nng thy gii DNA mach di nhng v[
tr xc d[nh
Vi khun s dng restriction enzyme d
bo v chng khi cc DNA ngoai Iai
Vi khun c mt co ch d bo v DNA
ca chng khi hoat dng ca cc
restriction enzyme ca bn thn.
Cc RE hp thnh h thng bo v
prokaryote, chua c h thng tuong t
no duc pht hin eukaryote.
Chng duc phn Ip v s dng cho cc
phng th nghim
Vai tr sinh hoc ca RE
H thng bin di gii han-restriction enzyme
duc hoat dng cng vi h thng methyIase.
MethyIases I enzyme thm nhm methyI vo
nucIeotide chuyn bit (vo A hay C) trong
trnh t nhn bit (recognition sequence). S
methyI ha ngn chn s nhn bit ca
restriction enzyme.
Do d, restriction enzyme trong mt t bo
khng phn ct DNA ca chnh n. Tuy nhin
restriction enzyme c th phn ct DNA ngoai
Iai xm nhp vo trong t bo nhu ca
bacteriophage.
Restriction Endonucleases
Loai I- c nhiu tiu don v[,c hoat tnh
endonucIease v methyIase, ct mt cch
ngu nhin tai v[ tr cch v[ tr nhn bit
(recognition sequence) 1000 bp
Loai II- ct DNA ngay v[ tr nhn bit, khng
cn ATP, hu ht dang don phn
Loai III- c nhiu tiu don v[, c hoat tnh
endonucIease v methyIase ct cch 25 bp
t recognition sequence
2
Restriction Enzyme
Restriction enzyme
(endonuIease) ct
DNA tai trnh t dc
hiu
Nhng Ioai cu ni
no b[ RE ct?
Cu ni dng ha tr[
(trong mt chui)
Cu ni hydrogen
(gia hai chui)
Cu ni
hydrogen
Cu ni dng ha tr[
RE hoat dng nhu th no?
Vj tr nhn bit ca enzyme
Mi enzyme ct DNA tai mt
trnh tu dc bit restriction
site
Enzyme nhn bit 4-, 6- hoc
8- cp base, trnh tu lp do
(palindromic sequence)
Hoat dng ca RE

Trnh tu dc hiu

Ct

5' AGCT 3'

3' TCGA 5'
Arthrobacter luteus Alu
5' GGATCC 3'
3' CCTAGG 5'
Bacillus
amyloliquefaciens
BamH
5'AAGCTT 3'
3'TTCGAA 5'
Haemophilus influenzae Hind
5' TCGA 3'
3' AGCT 5'
Thermus aquaticus Taq
5' GAATTC 3'
3' CTTAAG 5'
Escherichia coli EcoR
Trnh t nhn bit Ngun t vi sinh Enzyme
Cc RE ph bin
EcoRI
Escherichia coli
5' nh ra
HindIII
Haemophilus influenzae
5' nh ra
PstI
Providencia stuartii
3' nh ra
5'
CTGCAG
3'
3'
CACGTC
5'
5'
GAATTC
3'
3'
CTTAAG
5'
5'
AAGCTT
3'
3'
TTCGAA
5'
3
u dnh v du bng
Khi cc enzyme ct, chng c th tao
ra
u dnh: mt s RE, v[ tr ct Ich nhau
trn hai mach. Trong trung hp ny
cc du dnh c th bt cp tr Iai
u bng: mt s RE ct hai mach DNA
tai cng mt dim, sau khi ct hai du
bng khng c kh nng t kt hp Iai.
ni chng Iai phi dng enzyme T4
Iigase.
Trnh tu lp do
Enzyme ct
5'
GAATTC
3'
3'
CTTAAG
5'
5'
G
3'
3'
CTTAA 5'
5' AATTC
3'
3'
G
5'
Tao ra du 5' nh ra (theo hung 5')
u bng: qu trnh ni sau ny
khng dc hiu
5' GAA TTC 3'
3' CTT AAG 5'
HaeIII
Hae l mt restriction enzyme d trn
phn t DNA cho dn khi tm thy
mt trnh tu ca 4 base




Khi trnh t xc d[nh duc tm ra
HaeIII s ct DNA


Nhng sn phm ny duc gi
vi tn "du bng" = "blunt ends


4
Tn ca restriction enzyme dn t:
Loai vi khun m enzyme duc tm thy
Th t m restriction enzyme duc xc d[nh v
phn Ip.
la mL vl du
Chng ca vl khun
la resLrlcLlon enzyme du Lln ca L.coll duoc
khm ph.
ng dng RE
Vic s dung RE c ngha quyt djnh
trong su pht trin ca sinh hoc phn t
eukaryote. Chng cho php ct nh b
nhim sc th khng l eukaryote
Cc RE ch yu duoc s dung trong vic
tao dng vi muc dch tao ra mt s luong
ln cc bn sao ca mt trnh tu DNA xc
djnh.
Chng cng duoc ng dung d lp bn d
ct gii han (restriction map) vo vic
phn tch so snh b gen cc loi khc
nhau thng qua k thut RFLP (Restriction
Fragments Length Polymorphism Tnh
da hnh kch thuc ca cc trnh tu gii
han)
Bn d enzyme ct gii han
5
Cc enzyme thng dng khc
DNA poI xc tc s tng hp DNA theo chiu
5' 3' nhu:
DNA poI I (KIenow poI): poIyIerase 5' 3' , exonucIease 3' 5'
T4 DNA poI: ging KIenow poI nhung exonucIease 3' 5'
manh hon
Taq poI: phn Ip t Thermus aquaticus
Pfu poI : phn Ip t Pyroccus furiosus
Reverse transcriptase:do cc Retrovirus sn sinh
DNA terminaI transferase: Ii trch t tuyn c b
RNA poI tham gia vo vic tng hp RNA theo
chiu 5' 3', thng dng nht I:
SP6 RNA poIymerase: c ngun t phage xm nhim
SaImoneIIa typhimurium
T3 v T7 RNA poIymerase: c ngun gc t phage xm nhim
E.coIi
Polymerase
Cc ligase
y I enzyme xc tc cho phn ng
ni gia hai doan DNA (DNA Iigase)
hay RNA (RNA Iigase).
C cc Iigase sau:
T4 DNA (RNA) Iigase: Ii trch t phage T4
xm nhim E. coli
T4 poIynucIeotide kinase: Ii trch t phage
T4 xm nhim E. coli
AIkaIine phosphastase: Ii trch t E. coIi
hay t rut b
Cc nucIease
y I nhm phn ct DNA hay RNA.
Gm cc enzyme chnh sau:
DNAase I: c ngun gc t ty tang b
NucIease S1: Iy trch t Aspergillus
oryzae
ExonucIease III: tch chit t E. coli
RNAase A: hin din khp mi noi
RNAase H: dng trong tng hp cDNA
Tao dng phn t
(moIecuIar cIoning)
- Tao dng phn t dng k thut di truyn phn Ip
v Im thun mt gen:
+ Tch v phn doan DNA ngun
+ Gn cc doan DNA b[ ct vo vector dng ha
+ Bin nap v gi DNA ti t hp trong t bo ch: ngn
hng DNA (DNA Iibrary, DNA bank)
+ Pht hin v Im thun dng mc tiu
+ Nui cy dng mc tiu d Iy trch v nghin cu DNA ti
t hp
6
Ti t hp DNA
Ti t hp DNA s dng
restriction enzyme
Quan trng: ni hai
mnh DNA Iai vi nhau
chng phi duc ct
bng mt Ioai enzyme
ct gii han
Tai sao?
Cu hi.
Loai cu ni no do DNA Ligase tao
ra?
Cu ni dng ha tr[ (gia nucIeotide
trong mt chui DNA)
Restriction enzyme v
DNA Iigase c th duoc
s dung d tao DNA ti t
hp, DNA c th duoc ct
ni lai vi nhau t cc
ngun khc nhau
Tao dng (CIoning) c
ngha l gn mt doan
DNA (mt gen vo mt
vector tao dng (cIoning
vector) sau d dt vo mt
t bo sng ("Bin nap")
d khuch dai gen muc
tiu.
DNA CIoning
Khuch dai cc doan DNA
Ngun DNA d tao dng
DNA nhim sc th (bao gm DNA
"duoc dng v "khng duoc dng)
mRNA duoc chuyn thnh cDNA
PCR- khuch dai DNA muc tiu
S dng cc DNA duc tao dng I cch du tin xy
dng bn d ct gii han
phn tch gii trnh t DNA duc tao dng
S dng DNA tao dng Im probe xc d[nh r mc
d phin m, kch thuc ca mRNA
S dng DNA tao dng nhu mt probe d xc d[nh
DNA tuong t trong cc Ioi khc nhau hoc tao thnh
thu vin tao dng
Sn xut tht nhiu gen duc tao dng (V d
insuIin protein)
S dng tao dng d tao antisense mRNA cho gene
siIencing
Tao mt reporter gene d xc d[nh s biu hin ca
gen
Tai sao phi tao dng DNA?
Restriction endonucIease d ct DNA
DNA Ligase d ni DNA
Vector d gn DNA
T bo ch d khuch dai DNA
Cng c d chuyn DNA ti t hp vo t bo
ch
Cng c d gi DNA trong t bo ch
Cng c d xc d[nh xem t bo no d duc
tao dng thnh cng (tm sot)
Cng c tao dng
7
Th mang gen: vector
Mt vector cn c cc dc dim tiu chun sau:
C kh nng t sao chp trong t bo ch, sao chp
khng ph thuc vo t bo ch
C dc tnh gip d xc d[nh t bo c chng
Mang nhng v[ tr nhn bit duy nht ca cc
enzyme ct gii han
C kch thuc cng nh cng tt, d xm nhp, sao
chp nhanhhiu qu.
C s Iung bn sao trong t bo cao
Cc vector cn biu hin th phi c promoter manh
Th mang gen: vector
Vector: phn t DNA kch thuc nh dng d
mang gen, sao chp, thao tc trn gen
Vector tao dng v vector biu hin
Vector tao dng: chuyn v Iuu tr gen ti t
hp trong t bo ch, nhn bn, thao tc, phn
tch sau d trn DNA ti t hp s d dng hon
Vector biu hin: tao sn phm ca gen ti t
hp mc phin m, d[ch m, phn tch trnh
t diu ha s biu hin ca gen
S khc nhau ca cc vector tao dng
Loai t bo m chng c th vo
Cch thc m chng xm nhp (bng bin nap
hay bng cch xm nhim ca virus/phage)
In ca doan DNA ngoai Iai m chng c th
mang
V[ tr ct gii han chnh xc ca enzyme ct gii
han
S n d[nh ca DNA khi duc chn vo
Nhng kha canh quan tm
Hiu qu tao dng
S luong bn sao (trn mt t bo ch)
Kh nng tm sot (v du h thng Lac Z)
PIasmid I cc vector tao dng
Vj tr ct han ch duy nht d chn DNA muc tiu
(vng poIyIinker), c th nm ct ngang gen lacZ m
ha cho Beta-galactosidase, mt enzyme chuyn
khun lac thnh mu xanh khi hin din X-gal (khun
lac chuyn thnh mu trng khi c mt doan DNA
ngoai lai chn thnh cng vo vng polylinker v lm
bt hoat gen lacZ.)
C promoter manh nm hai pha ca vng
polylinker d biu hin DNA duoc tao dng.
Mt oriC
Marker chn Ic (V du. Gen khng khng sinh) nu
khng c bin nap vi khun s cht (chc chn rng
chi c nhng vi khun duoc bin nap thnh cng mi
sng)
8
PIasmid I nhng doan DNA ngn (2-5kb) dang
chui xon kp, dang vng, c th t nhn di dc
Ip vi DNA b gen
C s bn copy cao trong t bo E. coli (100)
Gen khc khng sinh (marker chn Ic, VD. Amp
R
;
c ngha I E. coli c kh nng khng khi bin nap
thnh cng nhn duc pIasmid)
Tao dng tt vi doan DNA 5-10 kb (nh)
Rt d s dng
PIasmid
Cc Ioai pIasmid
PIasmid th h th nht: tm thy trong tu
nhin ( ColE1, pSC101.)
PIasmid th h th hai: plasmid nhn tao
(pBR322: kch thuc 4364 bp, mang hai gen
Ap
R
, Tet
R
v 20 vj tr nhn bit duy nht ca cc
enzyme ct gii han)
PIasmid th h th ba: dy l cc plasmid
manh hin nay c hai dc tnh co bn
Kch thuc nh
C mang mt polylinker
9
Vector tao dng I phage
+ Bacteriophage vector
uc thit k d di vo chu trnh tan
Tao dng tt vi nhng doan DNA In (10-23 kb)
Phage khng d thao tc nhu pIasmid
Phage duc Ioai b 1/3 b gen chi gi Iai vng cha gen xm
nhp, t sao v Ip ghp
DNA ngoai Iai duc dua vo t bo ch qua phage ny, nhn bn
v Iuu tr cng vi phage
Vector phage cha t trnh t nhn bit ca enzyme ct gii han
+ Thc khun th M13
+ B gen mach don c kch thuc 6,4 kb
+ Thung dng d gii trnh t DNA
+ Phagemid
Kt hp gia bacteriophage v pIasmid
Tao ra + dng gi ssDNA vi s tr gip ca
phage
Hoc c th bin nap vo E. coli khi n th
hin ging mt pIasmid (kch thuc han ch
ging mt pIasmid)
V d. pBIuescript (pBS)
C promoter phage T3 v T7 trn mi hung
cho vic tng hp ssRNA in vitro
10
Cc vector tao dng khc
+ Cosmid
Thit k nhn tao bi s kt hp duc uu dim
pIasmid DNA v trnh t cos t phage
Khi vo trong t bo ch, c kh nng sao chp
nhu pIasmid. PIasmid ti t hp bn hon trong t
bo ch
Nhung trnh t cos cho php dng gi DNA vo
phage d ti nap vo t bo khc
Rt tt d tao dng nhng doan gen rt In
DNA (32-47 kb). Hiu sut chuyn gen cao hon
PIasmids cng In th cng kh thao tc
BAC
Nhim sc th nhn tao vi khun
Dng d tao dng nhng doan DNA rt ln vo E. coli
(tt v n djnh s dung trong tao dng HGP)
C cha OR ca nhn t f; s dung ging plasmid
Eukaryotic vector
VD. Ti pIasmid ca Agrobacterium tumefaciens dng
d tao dng thuc vt.
VD. YAC (Nhim sc th nhn tao nm men) Kch
thuc 10kb mang duoc DNA 150 1000kb. Sao chp
nhu mt nhim sc th bnh thung ca nm men
dng d tao bn d vt l b gen v du b gen ngui
(nhung khng bn nhu BAC)
11
Uu dim ca Prokaryote
1. Pht trin nhanh
2. Thao tc d dng
Nhuc dim ca Prokaryote
1. Khng th tch cc intron ra
2. ntron cn thit cho su biu hin ca gen
3. Kch thuc DNA dua vo vi khun bj han ch
4. Prokaryote khng c co ch glycosyl ha
protein
T bo ch
- E. coli:
+ T bo ch ph bin nht, d nui, sinh sn nhanh
+ Vi sinh vt gy bnh co hi
+ Khng tit enzyme
- Bacillus subtilis:
+ Khng gy bnh, khng tao ni dc t, tit protein
+ Pasmid khng n d[nh trong t bo ch
- Saccharomyces cerevisiae:
+ T bo eukaryote duc nghin cu di truyn k nht
+ S dng d th hin gen eukaryote
- Virt dng vt (SV40, retrovirus.):
+ Dng d dng ha gen vo t bo hu nh, co ch
tim tan
Ci tin t bo ch E. coIi
Thng thung E. coIi dng d tao dng
thung duc ci tin:
Loai b cc h thng sa di han ch
bng cch gy dt bin trong h thng
sa di han ch ni sinh
H thng ti t hp DNA duc sa di d
ngn chn s ti t hp
Thay di hoat tnh endonucIease nhm Im
tng Iung pIasmid tch Iy trong t bo
Cc buc ca k thut tao dng
1. C Ip gen mc tiu, c Ip pIasmid DNA
2. Thao tc trn DNA
a,b. Ct - Restriction enzyme
c. Ni - DNA Iigase
3. Bin nap vo vi khun
4. Chn Ic vi khun d bin nap thnh cng.
5. Xc d[nh dng vi khun mang gen cn tao
dng
12
Qu trnh tao
dng mt gen
ngui trong
mt vector tao
dng I
pIasmid vi
khun c th
duc chia
thnh 5 buc
Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Vector tao dng din hnh
Antibiotic resistance important to help select for
organisms that are transformed with the plasmid
olyclonlng slLe
Cen khng khng
slnh
Cen khng khng slnh. nu
chen vao mL gen s dung sL
l resLrlcLlon enzyme, kL qu
L bao khng LeLracycllne
nhung khng khng amplclllln
reslsLanL. 1al sao?
Phuong php khuch dai v thu nhn gene t vi khun vi pIasmid
Cen dlch
S dung cung mL loal resLrlcLlon endonuclease d cuL" clonlng vecLor va
Lhe hosL chromosome. 1al sao??
Phuong php khuch dai v thu nhn gene t vi khun vi pIasmid
Chn DNA ngoai lai
Bin nap
Phuong php khuch dai v thu nhn gene t vi khun vi pIasmid
Hin din cloning vector cho php chon loc trn agar c cha khng sinh
. Cc t bo khng cha cloning vector th khng c tnh khng khng sinh
13
PIasmid ti t hp
Tao plasmid ti t hop
Bin nap plasmid
vo E. coli
Ti tao dng (subcIoning)
Ti tao dng l kiu don gin nht ca th
nghim tao dng, l qu trnh chuyn DNA
d duoc tao dng t vector ny sang
vector khc d biu hin n trong mt
chng ch nht djnh.
Cc buc co bn ca ti tao dng cng
tuong tu nhu tao dng
Thu vin DNA
Da vo bn cht ca DNA cn tao
dng ngui ta phn bit hai Ioai thu
vin gen:
thu vin b gen v thu vin cDNA
14
Thu vin b gen (Genomic)
Thu vin b gen ca mt sinh vt I tp hp
tt c cc trnh t DNA cu thnh b gen d
duc gn vo vector. Cc vector ti t hp
sau d duc dua vo t bo ch. Cc t bo
ch sau d duc nui cy trn mi trung v
tao thnh cc dng.
Thu vin b gen thung duc Ip cho
prokaryote
B suu tp cc dng vi t nht mt bn sao
cho mi doan DNA duc tao dng trong YAC
hay BAC
Thu vin cDNA
Thu vin cDNA I tp hp cc bn sao cDNA t
tt c cc mRNA ca mt t bo. Nhu vy khng
ging vi thu vin b gen , thu vin cDNA duc
thit Ip t mt t bo xc d[nh trong d gen cn
nghin cu phi biu hin thnh mRNA
Thung duc Ip cho eukaryote
cDNA duc tng hp t mRNA trung thnh
(eukaryote) (Khng c cc doan intron) s dung
enzyme Reverse transcriptase, RNase H , DNA
PoIymerase I, v nucIease S1.
Thu vin b gen
15
Thu vin cDNA
Tuyn chn dng mc tiu
C th s dng mt khng th duc dnh
du d xc d[nh protein duc chn
t mt vi khun Iac vo mng Iai; ph
v t bo
Mu d trn mng Iai vi mt khng th
dc hiu cho protein chn
Hoc s dng mt mu d DNA (probe) b
sung d tm trc tip DNA
16
Tuyn chn dng mc tiu
c th phn Ip dng t bo ch ti t
hp cha gen mc tiu trong mt thu vin
DNA hay mt thu vin cDNA, ngui ta phi
s dng cc phuong php sng Ic.
Thng thung cc phuong php sng Ic
duc xy dng da trn vic s dng mu
d DNA.
Mu d DNA (probe) I mt doan
oIigonucIeotide duc dnh du v n b
sung hoc b sung mt phn vi gen mc
tiu.
Tuyn chn dng mc tiu
- Gen duc biu hin trong t bo
ch I vi khun:
+ Lai min d[ch (Western bIotting,
ImmunobIotting)
+ Hoat tnh enzyme
+ B tr dt bin (gen ngoai Iai dng
dang vi gen ca t bo ch)
- Gen ngoai Iai khng biu hin trong
t bo ch:
+ Lai insitu:
+ Lai khun Iac (coIony
hybridization):
vector I pIasmid
+ Lai pIaque (pIaque coIonization):
vector I phage
Vector biu hin (expression vector)
- biu hin gen ngoai Iai:
+ Thu protein ti t hp
+ Nghin cu cc yu t diu ha th hin ca gen
- Gen ngoai Iai duc kim sot bng promoter duc
nhn din bi RNA poIymerase ca t bo ch:
+ Promoter manh
+ Khung d[ch m dng
+ Promoter thung dng: lac, trp (co ch tt m)
17
ng dng thc tin ca
k thut di truyn
- Sn xut nhng sn phm trao di cht ca vi
sinh vt
- Sn xut vcxin phng chng virus
- Sn xut protein ca dng vt hu nh
- Tao ra cc thc vt, dng vt chuyn gen
- Phn Ip v ng dng ngun gen vi sinh trong
x I mi trung
- Tao cc thuc diu ha s th hin ca gen
- Liu php gen cha tr[ cc bnh di truyn
ng dng ca Sinh hc phn t
Cng ngh tao cc sinh vt mi c ch.
Nhng nghin cu ch yu tp trung
vo vic:
Nng cao nng sut nui trng
Tao ra cc th h dng vt mi
C thm mt s tnh trang mi
Nhiu trng hon
T I nac cao hon
San pham cong nghiep
Trong nng nghip
Cng ngh sn xut ging cy trng, vt
nui vut gii han ca tin ha d chng
ch[u bnh, thch nghi vi cc diu kin sinh
thi v cho nng sut cao.
Cng ngh sn xut cc Ioai thc phm
mi c gi tr[ dinh dung cao, c gi tr[
thuc - dinh dung, cc cht b sung
(cc vitamin..) cc protein don bo.
18
T nhng nm 1970, cc nh khoa hc
d c th chuyn mt gen Ia vo vi
khun v bt n biu hin gen
Tuy nhin, nhng protein phc v cho
nhu cu ca con ngui ngy cng di
hi phc tap v co th vi khun khng
biu hin duc v cn dn dng vt
chuyn gen
Sn phm thnh cng du tin ca con ngui
v sn xut sn phm sinh hc thng qua
chuyn gen I InsuIin v hormon sinh trung
vo vi khun (1982 v 1987)
Nm 1998 c khong dui 1% duc
phm I protein duc tng hp ti t
hp nhung tr[ gi ti 12 t USD
Ngui ta d tnh chi cn 600 con b
chuyn gen I c th cung cp d nhu
cu ca c th gii v mt duc phm I
mot protein no d (v d human Serum
aIbumin diu tr[ bng)
Trong Y hoc
Tao ra cc Ioai protein, hormon, yu t sinh trung
dng trong tr[ Iiu (ch yu trong sa v trong
huyt thanh)
Thay di cu trc gii phu, sinh I co quan, ni
tang (dua mt s gen mi, Ioai b mt s gen)
nhm phc v mc dch cy ghp ni tang.
Tm hiu cc gen gy bnh nhu gen gy Ioan
dung co tim, ung thu, min d[ch, hng cu Iui
Iim.
Dng phuong php chuyn gen d tao dng vt th
nghim m b[ Ioai b hoc kho cc gen no d d
xc d[nh chc nng ca gen.
Liu php gen v Iiu php thay th t bo v m.
19
Phuong php chun don bnh mi
K thut tao dng(cIoning) v dc bit
I k thut PCR dua dn k thut chun
don mi bng Iai DNA v RNA.
Phuong php ny hin dang duc ng
dng rng ri trong chun don cc
bnh di truyn, quan h dng h, ti
pham hnh s..
Mt s thnh tu Vit Nam
1997, hon thin quy trnh cng ngh
chuyn gen hormon sinh trung ngui
vo c vng (Carassius auratus)
Nm 2003, Vin Sinh hc nhit di d
chuyn gen Bt khng su vo cy
thuc I (Nicotiana tabacum) v cy
ng (Zea mays)
Nm 2005, Vin Sinh hc nhit di d
chuyn gen hormon sinh trung ngui
vo c chp (Cyprinus carpio)