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S chuyn gen thc vt dng A.

tumefaciens da trn nguyn l nh hnh sau:

1. Chuyn gen

Hnh: Agrobacterium mang plasmid gy khi u thc vt (tumour inducing plasmid- Ti


plasmid), plasmid ny cha cc gen vir v vng gen cn chuyn (T-DNA). Gen quan tm c chn vo vng T-DNA. Cc t bo tn thng thc vt tit ra hp cht bo v, hp cht ny kch thch s biu hin gen vir Agrobacterium. Vir protein to ra T-strand t vng T-DNA trn Ti-plasmid. Sau vi khun gn vo t bo thc vt, T-strand v mt s protein vir chuyn vo t bo thc vt qua knh vn chuyn. Trong t bo thc vt, cc protein vir tng tc vi T-strand to ra phc h (T- complex). Phc h ny vo nhn t bo thc vt, T-DNA chn vo b gen thc vt v biu hin gen quan tm. (Nature 433, 583584 (2005))

Cho n nay ngi ta ch dng mt loi vi khun Agrobacterium tumefaciens chuyn gen nhng mt cng b mi y trn tp ch Nature (ngy 10 thng 2 nm 2005), Broothaerts v cng s tm ra cch chuyn gen thc vt dng 3 loi vi khun mi (1). Trong cng trnh ca h, tc gi dng 3 chng Rhizobium loi NGR234, Sinorhizobium meliloti v Mesorhizobium loti th nghim chuyn gen vo mt s loi thc vt khc nhau thuc hai nhm thc vt bc cao: nhm mt l mm v nhm hai l mm. C ch chuyn gen ca 3 loi vi khun ny v thc vt ging vi c ch chuyn gen ca Agrobacterium. Hiu qu chuyn gen ca 3 vi khun ny tng i thp, t 1 n 40% so vi Agrobacterium. Tuy nhin tc gi cho rng nu thay i mt s nhn t nh tui v loi m thc vt c th nng cao hiu qu chuyn gen. Tn sut chuyn gen cao ca Agrobacterium l kt qu ca qu trnh nghin cu lu di t hn 20 nm nay. Cng trnh ca Broothaerts v cng s rt c ngha cho ngnh cng ngh sinh hc thc vt. Kh nng chuyn gen ca mt s loi vi khun sang thc vt c th ng gp vai tr quan trng trong s tin ha ca thc vt.

Dng A. tumefaciens chuyn gen cc nh khoa hc gp mt s kh khn v lut bn quyn v iu ny lm cho gi thnh tng cao gy kh khn cho nghin cu cng ng v cc nc thuc th gii th 3. Trng nhm nghin cu Richard A. Jefferson ni rng k thut ny s c ng dng rng ri v n c CAMBIA cp bng sng ch. CAMBIA l mt t chc phi li nhun s hu nhiu sng ch theo

nguyn tc ngun m. CAMBIA s cho php k thut ny c p dng rng ri ngn chn mt s cng ty ln thao tng.

Chuyn gen (transgenesis) l a mt on DNA ngoi lai vo genome ca mt c th a bo, sau on DNA ngoi lai ny s c mt hu ht cc t bo v c truyn li cho th h sau. V vy khi nim chuyn gen ch c s dng cho thc vt v ng vt. Nm men, vi khuNn v t bo nui cy mang mt on DNA ngoi lai c gi l cc t bo ti t hp (recombinant cell) hoc t bo bin np (transformed cell). Chuyn gen khc vi liu php gen (gene therapy). C trng hp cc t bo mm khng mang DNA ngoi lai. Thut ng liu php gen mm (germinal gene therapy) cng c s dng. Liu php gen mm hy cn cha c th nghim ngi. Cc t bo mm ny mang DNA ngoi lai v c truyn li cho th h sau. V mt lch s, thut ng GMO (genetically modified organism)-sinh vt bin i gen, c s dng ch yu ch cc thc vt chuyn gen c gieo trng cung cp lng thc, thc phm cho con ngi v ng vt. Logic hn v chnh xc hn, GMO cp ti tt c cc c th sng bin i di truyn, bao gm c vi sinh vt. Thut ng GMP (genetically modified plant)-thc vt bin i gen v GMA (genetically modified animal)- ng vt bin i gen cng c s dng. Trong thc t, cc on DNA ngoi lai c s dng to sinh vt chuyn gen hu ht l cc gen lun c sn mt trnh t ph hp vi mt promoter lm cho n biu hin thnh RNA, ni tng qut l protein. Sn phm phin m ca gen c th l mt RNA khng c dch m thnh protein. y l trng hp i vi RNA ngc hng (antisense RNA), rybozyme v cc gen c phin m bi RNA polymerase I v III. Khng nht thit l DNA ngoi lai lun lun c hp nht vo genome ca sinh vt chuyn gen. DNA ngoi lai khng th tn ti trong c th m khng hp nht vo trong genome ca n. Mt on DNA t do nhanh chng b loi tr trong chu trnh t bo v vy n s khng c kh nng ti bn v truyn li cho cc t bo con. Tuy nhin v l thuyt th c th duy tr mt on DNA ngoi lai nh mt nhim sc th nh (minichromosome) c kh nng t ti bn v c mt trong cc t bo con. Mt s genome virus c c tnh ny, v d nh virus herpes. Mt vi on nhim sc th thng c tm thy cc t bo khi u, l cc nhim sc th tn ti trong mt thi gian ngn, mang cc yu t ti bn v truyn cho cc t bo con.

2. ng vt (Thc vt) chuyn gen


ng vt (Thc vt) chuyn gen l ng vt (thc vt) c gen ngoi lai (gen chuyn) xen vo trong DNA genome ca n. Gen ngoi lai ny phi c truyn li cho tt c mi t bo, k c cc t bo sinh sn mm. Nu dng t bo mm b bin i, cc tnh trng b bin i ny s c truyn cho cc th h k tip thng qua qu trnh sinh sn bnh thng. Nu ch c dng t bo sinh dng b bin i, ch c c th mang cc t bo sinh dng

b nh hng v khng di truyn li cho th h sau. Vic chuyn gen ngoi lai vo ng vt (thc vt) ch thnh cng khi cc gen ny di truyn li cho th h sau. Cho n nay, trn th gii ngi ta thnh cng trong vic to ra nhiu thc vt, ng vt chuyn gen. ng vt, khng ch i vi ng vt m hnh (chut), vt nui (b, ln, d, cu, th, g, c...) m c nhng loi ng vt khc nh kh, mui v mt s cn trng...

3. Gen chuyn
Gen chuyn (transgene) l gen ngoi lai c chuyn t mt c th sang mt c th mi bng k thut di truyn. Cc gen chuyn c s dng to ng vt, thc vt chuyn gen c ngun gc t cc loi sinh vt khc nhau: ng vt, thc vt, vi sinh vt v c con ngi. V d: gen ca ngi c a vo chut v cc vt nui khc nh ln, b, cu, chim...

4.Mc ch ca vic chuyn gen


Ni chung, mc ch ca chuyn gen l thm mt thng tin di truyn ngoi lai vo genome, cng nh c ch mt gen ni sinh. Trong mt s trng hp, s thay th mt gen hot ng chc nng bng mt gen hot ng chc nng khc l cn thit. Gen ngoi lai c th l mt th t bin ca gen ni sinh hoc mt gen hon ton khc. S thm gen c th c thc hin cung cp sinh vt mang protein mi. S thm gen cng c th c s dng nghin cu c ch hot ng ca mt promoter trong ton c th. S kt hp ca gen reporter vi promoter l nguyn tc chung ca phng php ny. S thay th gen c s dng ch yu lm bt hot mt gen bit. Trn thc t, n bao gm s thay th gen ni sinh bng mt th t bin bt hot. Phng php ny c dng cho thng tin v chc nng sinh hc ca gen nh trng hp thm gen. Thc vy c thm gen v bt hot gen c th gy ra cc bin i sinh vt chuyn gen, m cc bin i ny c th c quan st hoc nh gi. Cc th t bin ca gen thay th c th cung cp thng tin bng mt cch thc tinh vi hn. S thay th mt gen bng mt gen c chc nng khc nhau hon ton l kh hn nhng c th thc hin a mt marker hoc mt gen chn lc vo genome. V tr hp nht vo genome c th c chn lc i vi kh nng cha ca n biu hin gen ngoi lai bng mt cch chc chn.

5. Nguyn tc c bn trong vic to ng (thc vt) chuyn gen


Nguyn tc c bn trong vic to ng vt (thc vt) chuyn gen l a mt hoc vi gen ngoi lai vo ng vt (thc vt) (do con ngi ch ng to ra). Cc gen ngoi lai ny phi c truyn thng qua dng mm v vy mi t bo k cc t bo mm sinh sn ca ng vt (thc vt) u cha vt cht di truyn c sa i nh nhau.

6.Cc phng php chuyn gen

Nhiu phng php khc nhau c pht minh a gen vo t bo v m ng vt v thc vt. K thut n gin nht l chuyn DNA trn bng vi tim (microinjection), xung in(electroporation), sng bn gen. Cc phng php vector virus, t bo gc phi, chuyn gen gin tip nh vi khun phc tp v hiu qu hn bao gm s dng cc phc hp lipid-DNA (liposome), Agrobacterium, chuyn gen bng sng bn gen... Tu thuc vo i tng chuyn gen m ngi ta la chn phng php chuyn gen ph hp.

6.1.T bo ng vt
6.1.1. DEAE-dextran DEAE-dextran l mt polycation. y l tc nhn ha hc u tin c s dng chuyn DNA vo t bo ng vt nui cy. Trong phng php ny, DNA ngoi lai c cho vo mt trng nui cy vi s c mt ca DEAE-dextran. DEAE-dextran s kt hp vi DNA tch in m (Hnh 2.1). S d tha in tch dng trong phc hp DNA-polycation do s ng gp ca polycation, cho php phc hp ny i n kt hp cht ch hn vi mng t bo tch in m. S xm nhp ca phc hp c th t c nh s nhp bo (endocytosis).

Hnh 2.1: S kt hp gia DEAE-dextran v DNA Phng php ny c s dng thnh cng trong vic chuyn DNA vo cc t bo biu hin nht thi, c ngha l thi gian biu hin ngn ch vi ngy trong qu trnh nghin cu. Tuy nhin, phng php ny ni chung l khng hu ch i vi cc nghin cu cn s chuyn nhim n nh da vo s hp nht ca DNA ngoi lai vo nhim sc th, hiu qu bin np thp, ch c s dng cho mt s loi t bo v cho kt qu tt i vi t bo ex vivo. Cc polycation khc cng c s dng chuyn DNA vo t bo nh polybrene, polyethyleneimine v dendrimers. 6.1.2. K thut calcium phosphate

K thut calcium phosphate (calcium phosphate technique) c pht trin u tin l xc nh s ly nhim ca DNA virus (Graham,1973) v hin nay c s dng rng ri th nghim hot ng bin np ca DNA virus cng nh DNA tch chit t cc t bo eukaryote (Wigler, 1978; Graham, 1979; Pellicer, 1980). K thut ny yu cu cc t bo nhn vi cc cht ng kt ta DNA v calcium phosphat (Hnh 2.2). Kt ta ny bm vo t bo v sau s hp th vo t bo qua qu trnh m bo (Loyter, 1982). Trong t bo, cc phn t DNA ngoi lai nm trong khng bo c to thnh do m bo v lysosome th hai nhng rt t DNA i n nhn v hp nht vo genome ch.

Hnh 2.2: Phc hp DNA-calcium phosphat Cho n nay, y l k thut v cng c gi tr i vi cc nghin cu chuyn gen vo cc t bo soma nui cy v ang c s dng nhiu chuyn cc dng genome vo t bo ch. T l cc t bo c bin np n nh ca k thut ny l tng ng vi phng php vi tim nhng khc vi vi tim l nhiu t bo c bin np cng mt ln. Hn na, bin np DNA tch chit t cc t bo ung th vo cc t bo nhn khng ung th cho thy y l phng php duy nht nghin cu s kim sot di truyn ca ung th. Phng php ny c s dng ph bin bi v n gin, protocol d thc hin, t tn km, s t bo cht sau bin

np khng ng k, s biu hin gen c th l nht thi hoc n nh v quan trng trong vic thit k vector virus ti t hp. Tuy nhin hiu qu bin np v mc biu hin ca gen chuyn thp. 6.1.3. Phng php vi tim S thnh cng u tin trong nghin cu to chut chuyn gen bng phng php vi tim vo tin nhn c cng b vo nm 1980 (Gordon, 1980). Mc du cu trc t hp gen chuyn c chng minh l tch hp vo genome ca chut, n c sp xp li nhng gen ngoi lai khng c biu hin. Cc cng b tip theo (Brister, 1981; Costantini v Lacy; 1981) chng minh rng vi phng php vi tim, cc gen chuyn tch hp v c kh nng biu hin. Vo nm 1982, ln u tin s thay i kiu hnh c th nhn thy chut nht chuyn gen c m t (Palmiter, 1982). y l kt qu biu hin ca gen hormon sinh trng chut cng chut nht. T n nay c rt nhiu cc cng trnh v chuyn gen, trong phn ln l cc nghin cu hiu qu ca gen vi tim vi s sinh trng ca ng vt c v v bnh hc. Nguyn tc ca phng php vi tim l mt lng nh DNA c tim trc tip vo nhn t bo phi trn hoc t bo nguyn vn mt cch c hc di knh hin vi. Phng php ny cho php a gen vo ng v tr mong mun tng t bo vi hiu qu tng i cao.Tuy nhin do i hi phi tinh vi, t m v cc k chnh xc nn hn ch s lng t bo vi tim v ngoi ra cn c th lm tn thng n t bo phi do tc nhn c hc gy ra khi tin hnh vi tim. bin np gen vo t bo bng phng php vi tim trc ht phi ch to kim tim v kim gi. Kim c to ra t nhng ng thu tinh do capillar ng knh 0,1-1,5 mm c si bng wolfram mnh trong nh h thng thit b lm kim. H thng ny gm c my ko kim t ng (pipette puller), my mi kim v my gia c kim.

Hnh 2.7: Vi tim gen ngoi lai vo tin nhn ca trng th tinh to kim tim trc ht phi to ra u kim nhn nh my ko kim. Sau s dng my gia c ct u nhn ca ng capillar sau khi ko to ra mi kim tim c ng knh thch hp. ng knh bn trong mi kim tim tu thuc vo loi t bo chuyn gen. i vi trng tin nhn ng vt c v c ng knh khong 70 m th ng knh ca kim tim khong 0,75 m l thch hp, i vi phi c mt t bo ng knh ca kim tim l khong 3-4 m. Cui cng a kim ln my mi lm sc nhn v trn lng u kim (Hnh 2.8). Kim gi c ch to t nhng ng capillar c u nhn bnh thng c nh trng trong qu trnh vi tim. to kim gi, dng my ko kim t ng to ra u kim nh sau dng bt kim cng hoc si platin t nng ct bt u nhn to ra u kim vi ng knh thch hp. Lm nhn u kim gi bng cch ng kim ny ngay trn mt on cong platin ca my gia c. t nng si platin v iu chnh u kim gi cho n chy t t. Quan st di knh hin vi v dng li khi u kim t yu cu. Vi tim c tin hnh trn h thng thit b vi tim (Hnh 2.9). H thng ny gm c hai b phn chnh l knh hin vi v my vi thao tc.

Hnh 2.8 : Cc my lm kim (Hng Narishige) 1. My gia c kim Microforge 2. My mi kim 3. My ko kim t ng Pipette Puller Knh hin vi dng cho mc ch ny l knh hin vi soi ngc (vt knh xoay ngc ln). phng i thch hp cho vic tin hnh vi tim vo phi c mt t bo l khong t 40-60 ln. My vi thao tc gm 2 phn ging ht nhau c b tr hai bn knh hin vi, mt dng iu chnh kim tim, mt dng cho kim gi. Tnh nng ca my ny l cho php iu chnh cc kim theo khng gian 3 chiu. Kim tim v kim gi c lp vo my vi thao tc v c ni vi syringe qua ng bng cht do c np y du parafin.Chun b dung dch gen chuyn: gen chuyn c xen vo trong mt vector plasmid v to dng trong E.coli. Cc vi khun bin np mang plasmid ti t hp c pht hin trong mi trng nui cy c thuc khng sinh do plasmid mang cc gen khng thuc c hiu. Cc vi khun sng st c sinh trng trong mi trng dinh dng thch hp v plasmid ti t hp mang gen chuyn s c sao chp mi khi t bo vi khun phn chia. Sau , hng triu bn sao ca plasmid ti t hp mang gen chuyn c tch chit t cc t bo vi khun ny v cc on gen chuyn c tch ra t plasmid ti t hp nh s dng enzym hn ch. tinh sch, gen chuyn c in di trn gel agarose. Ho tan gen chuyn trong dung dch m c trng (nh dung dch Tris-EDTA; dung dch TE...) v tnh nng dung dch gen chuyn nh quang ph k. Nng dung dch DNA s dng cho vi tim thng l 1-5 g/ml. Trc khi chuyn vo phi, gen chuyn c kim tra s biu hin trong cc t bo nui cy bng s chuyn nhim (transfection).

Hnh 2.9: My vi thao tc Olympus (Hng Narishige) 1. Knh hin vi soi ngc 2. My vi iu chnh Vi tim c tin hnh qua cc bc: np gen vo kim tim bng phng php capillar (ngm u kim tim vo dung dch gen khong 10-12 gi) hoc bm trc tip dung dch gen vo, lp kim tim v kim gi vo my vi thao tc, chuyn trng tin nhn vo a petri c cha mi trng c t di knh hin vi, gi trng tin nhn vo u kim gi bng lc ht ca syringe, iu chnh knh hin vi xc nh a phi v iu chnh my vi thao tc a kim tim vo v tr ca trng tin nhn, y gen vo trng tin nhn bng cch vn nh syringe. Khi thy trng tin nhn hi phng to v tr nn sng hn th dng li v ko nhanh kim tim ra. Trng tin nhn sau khi tim c di chuyn xa n cui a petri trc khi tim trng tin nhn tip theo. Mi mt nhm trng tin nhn hon thnh c chuyn sang mt a mi trng khc p v nh gi bng mt trong mt vi ting. Sau tt c cc trng tin nhn c nhn thy r rng v c chuyn vo ng dn trng ca con ci nhn. Cho n nay, trong cc k thut chuyn gen vo ng vt th phng php vi tim dung dch DNA vo hp t l phng php c hiu qu nht trn ng vt c v v hin l phng php ch yu c s dng chuyn gen vo vt nui. i vi thc vt th phng php ny c s dng i vi cc t bo tin phi ca hp t hoc cc t bo tin phi ca ht phn. Tuy nhin s xm nhp ca gen chuyn vo DNA t bo vt ch l mt qu trnh ngu nhin v xc sut gen chuyn xen vo v tr DNA vt ch m s cho php n biu hin l thp. Hiu qu ca vi tim l khng cao (Bng 2.1).

Bng 2.1: Hiu qu vi tim mt s loi ng vt Loi ng vt Th Cu Ln S lng trng c vi tim 1907 1032 2035 S lng con sinh ra t trng vi tim 218 (11,4%) 73 (7,1%) 192 (9,4%) S lng con chuyn gen 28 (1,5%) 1(0,1%) 20 (1%)

6.1.4. Chuyn gen qua liposome Vo thp nin 1980, liposome nhn to c s dng a DNA vo t bo. Lipid vi ton b li tch in dng pH sinh l l thnh phn lipid tng hp ph bin nht ca liposome c pht trin cho chuyn gen (Hnh 2.3). Thng th lipid cation c trn vi mt lipid trung tnh nh L-dioleoyl phosphatidylethanolamine (DOPE) (Hnh 2.4). Phn cation ca phn t lipid kt

hp vi DNA tch in m v kt qu l cha y DNA trong phc hp liposomeDNA (Hnh 2.5). i vi cc t bo nui cy, ton b li tch in dng ca phc hp liposome-DNA ni chung l gy ra hiu qu chuyn gen cao hn bi v n cho php phc hp ny kt hp vi mng t bo tch in m bn hn. Nh c ch nhp bo, cc phc hp xut hin trong endosome v sau i vo nhn (Hnh 2.6). Cha r DNA c phng thch t endosome v i qua mng nhn nh th no. DOPE c xem l mt lipid kch thch s dung hp v vai tr ca n l phng thch cc phc hp ny t endosome cng nh lm cho s dung hp ca mng t bo pha ngoi vi phc hp liposome-DNA xy ra d dng. Trong phng php ny, cc i phn t trc ht c a vo trong cc ti phospholipid. Cc loi ti khc nhau c m t, nhng ti mt lp mng l thch hp nht cho chuyn gen v chng c t l khong trng cha nc bn trong tng i cao i vi mi n v lipid v bi v chng c t l 59 phn phi cao hn. S dung hp ca liposome vi mng plasma l mt s kin him. Hiu qu bin np ca phng php ny thp hn so vi phng php vi tim vo tin nhn. Cc n lc nghin cu ang c tin hnh tm ra cc iu kin th nghim m c th lm tng s phng thch cc phn t kt nang t con ng m bo.

Hnh 2.3: Cu trc tng qut ca lipid cation tng hp

Hnh 2.4: Cu trc ca DOPE (L-diolecyl phosphatidylethanolamine)

Hnh 2.5: Phc hp liposome-DNA

Hnh 2.6: S hot ng ca vector liposome

Liposome c s dng a protein, lipid v cc phn t nh vo nhiu loi t bo nui cy, tuy nhin hiu qu thp hn vi tim i vi RNA hoc protein. Cng nh th, chuyn gen qua liposome v s biu hin ca gen chuyn l khng vt qua c cc phng php chuyn gen thng thng (nh h thng virus), s biu hin gen chuyn thng nht thi, s c ch bi cc thnh phn ca huyt thanh c th xy ra. Bn cnh , k thut ny c nhiu u im l gen chuyn s khng hp nht vo genome ch, c hiu qu tt i vi c t bo in vitro v in vivo, c th mang c cc DNA c kch thc rt ln, tinh khit cao, khng gy min dch, c th s dng vi cc t bo m bin np bng k thut calcium phosphate khng c hiu qu.. ng dng trong tng lai ca k thut ny ch yu s l kh nng phn phi thuc vo cc t bo ca c th sng. Hin nay k tht ny ang c pht trin ci tin s phn phi c hiu ca liposome bng cch ghp cc khng th c hiu vi b mt ca liposome ch. 6.1.5. Phng php chuyn gen nh vector virus Vo nm 1974, ln u tin cc nh khoa hc pht hin thy rng, sau khi tim DNA ca retrovirus SV40 vo khoang phi (blastocoel) ca ti phi chut, DNA ny c th c tm thy sau trong cc t bo ca chut trng thnh (Jaenisch,1974; Jaenisch v Mintz,1974). DNA provirus Mo-Mulv s dng trong cc th nghim nh th ny tch hp vo genome v truyn li cho th h sau, do to nn cc dng chut n nh (Stuhlmann, Jahner v Jaenisch,1981). T , vic s dng virus lm vector cho cc DNA ngoi lai c pht trin. Phng php ny tuy thao tc hi phc tp nhng c u im l hiu qu chuyn gen cao. Hn na gen cu trc gn vo vector virus s s dng promoter ca virus, cc promoter ny thng c tnh cao do gen cu trc ny s c biu hin mnh trong t bo ch. V nguyn tc, bt k loi virus no cng c th c s dng lm vector chuyn vt liu di truyn vo trong t bo. Nhiu nhm trong s , cc papovavirus, adenovirus, retrovirus...c s dng vo nhng mc ch chuyn bit. s dng lm vector, cc phn khc nhau ca genome virus c thay th bng gen cu trc quan tm. Virus c th c s dng ly nhim vo t bo giai on sm ca phi trc khi c chuyn ghp vo con m. Gen chuyn vi vector retrovirus xm nhp mt cch hiu qu vo h gen ca vt ch nhng virus s dng phi l virus an ton, khng gy bnh. Cc c th chuyn gen sinh ra t phng php ny l dng khm, c ngha l khng phi tt c cc t bo ca c th u mang retrovirus. Gen chuyn ch c th di truyn c nu retrovirus hp nht vo mt s t bo sinh dc. i vi phng php ny t l sng ca cc ng vt chuyn gen s sinh l rt thp. Nu nh cc thao tc di truyn l chun xc, khng gy ra s sy thai, th th h ng vt u tin (F1) cn kim tra s biu hin ca gen chuyn. Khi gen chuyn hp nht trong cc t bo sinh dc th c gi l th khm dng mm v sau chng c lai cng dng khong 10-20 th h cho n khi thu c cc ng vt

chuyn gen ng hp t v gen chuyn c mt trong tt c mi t bo. giai on ny, phi mang gen chuyn c th c ng lnh v c bo qun cho cc qu trnh cy chuyn v sau.

Hnh 2.11: Chuyn gen nh vector l virus 6.1.6. Chuyn gen bng cch s dng t bo gc phi Xut pht t l do cc t bo gc phi (t bo phi giai on 16-32 t bo) l cc t bo a nng (totipotent) ngha l c th phn ho thnh bt k loi m no v t s to nn c th hon chnh. T cc ti phi nui cy in vitro, ngi ta tin hnh tch chit cc t bo gc phi v bin np gen ngoi lai vo nhng t bo ny. Sau khi chn ra nhng t bo c bin np gen l ngi ta a n vo phi khc giai on phi nang to ra ng vt chuyn gen th khm. Trn 30%

ng vt chuyn gen to thnh l nhng ng vt chuyn gen th khm dng mm mang kiu gen ca dng t bo ny. y cc t bo gc phi c s dng nh l mt phng tin chuyn gen (Mintz, 1977). u im ca phng php chuyn gen ny l t l phi sng st sau thao tc, s tch hp v biu hin tnh trng ca gen mi kh cao. iu quan trng hn l trong thc t vic chuyn gen c th c tin hnh thng qua s thao tc vi phi du v ti phi. Phi cc giai on ny c th thu nhn m khng cn phu thut (c bit l i vi b), do vy cng vic chuyn gen c tin hnh rt d dng. Phng php ny c ngha c bit i vi s nghin cu kim tra di truyn ca cc qu trnh pht trin. S thun li ca n l cho php to ra mt cch chnh xc cc t bin gen xc nh bng ti t hp ng dng. Trong tng lai phng php s dng t bo gc phi s c s dng rng ri to ng vt chuyn gen. C ba cch to ng vt chuyn gen t cc t bo gc phi mang gen chuyn: - Th nht, phng php c dng trc mt l bm mt s t bo gc phi (khong 5-10 t bo) vo trong xoang phi nang ca t bo ng vt. - Th hai, xen mt s t bo gc phi vo gia bo thai thi k 8 t bo. - Th ba, nui cy chung t bo gc phi vi phi qua m.

Hnh 2.12: Phng php chuyn gen bng cch s dng t bo gc phi

6.2.Phng php chuyn gen vo t bo thc vt


Cc phng php a gene mi vo t bo thc vt c th chia lm 2 loi: phng php trc tip (phng php vt l ) nh bn gen hay in bin np (electroporation) v phng php gin tip (phng php t nhin), s dngAgrobaterium. Pht kin v vic Agrobaterium tumefaciens c kh nng chuyn gene vo thc vt bin loi ny tr thnh mt trong nhng cng c quan trong nht ca Cng ngh sinh hc thc vt. 6.2.1.Phng php gin tip 6.2.1.1. Phng php chuyn gen gin tip nh Agrobacterium A. tumefaciens l loi vi khun t c kh nng chn gene ca mnh vo thc vt v sau s dng b my thc vt biu hin cc gene ny di dng hp cht dinh dng cho chng. A. tumefaciens s to ra cc khi u thc vt gn im tip ni gia r v thn, gy ra nhng nt sn cy (bnh mn cy-crown gall disease). Agrobacterium c kh nng xm nhim t bo thc vt bng cch chuyn mt on DNA ca n vo t bo thc vt. Khi DNA vi khun c hp nht vi nhim sc th thc vt, n s tn cng vo h thng t chc ca t bo mt cch c hiu qu v s dng n m bo cho s sinh si ca qun th vi khun. Tht khng may mn cho cc nh trng cy n qu khi gp phi loi vi khun ny. Bi v n chnh l th phm gy ra bnh khi u hnh chp v bnh lng r nhiu loi cy cnh v cy n qu.

Mc d h thng chuyn gen gin tip nh Agrobacterium l c hiu qu i vi mt s loi nhng khng phi tt c thc vt c th c bin np bng con ng ny. c bit, lp mt l mm bao gm cc cy ng cc chnh trn th gii nh la, la m v ng l khng c bin np d dng nh A. tumefaciens. khai thc v s dng A. tumefaciens nh l mt vector chuyn gen cc nh khoa hc loi b cc gen gy khi u v gen m ho opine ca T - DNA v thay th vo l cc marker chn lc, trong khi vn duy tr cc vng b phi v b tri ca T-DNA v cc gen vir. Gen chuyn c xen vo gia cc vng b ca TDNA. N s c chuyn vo t bo v tr nn hp nht vi nhim sc th t bo thc vt Phng php chuyn gen gin tip nh Agrobacterium c kim tra i vi s xm nhp bn vng, s biu hin v s di truyn ca cc gen chuyn c bit. Tuy nhin, mt vi yu t nh hng n hiu qu bin np l loi m c bin np, giai on pht trin ca m, mc khi u ca vi khun A. tumefaciens s dng, mi trng nui cy m sau khi bin np, marker c s dng chn lc th bin np, loi vector s dng v kiu gen ca thc vt. 6.2.1.2. Chuyn gen nh virus v phage 6.2.2.Phng php trc tip

6.2.2.1. Chuyn gen trc tip vo protoplast


DNA d xm nhp c vo t bo thc vt, phi loi b vch t bo to protoplast. Protoplast c th c duy tr trong mi trng nui cy nh cc t bo sinh trng mt cch c lp hoc vi mt mi trng c hiu, vch t bo c th c to thnh v ton b cc cy c th c ti sinh t cc t bo ny. Qu trnh chuyn gen nh th ny c thc hin mt cch trc tip bng mt c ch vt l n gin, khng cn c vector. nng cao hiu qu bin np, ngi ta x l protoplast vi PGE (polyethylene glycol) hoc bng xung in. Phng php chuyn gen ny rt c hiu qu, c bit i vi nhng loi thc vt m phng php chuyn gen gin tip nh Agrobacterium khng th thc hin c. Tuy nhin, vic to protoplast cng nh ti sinh cy t protoplast khng n gin, tn nhiu cng sc, b nh hng ca nhiu yu t mi trng. Vi phng php ny, cc nh khoa hc chuyn gen thnh cng vo mt s loi cy mt l mm nh loi la ph Japonica (Datta, 1990), ng (Doon, 1990), la m (Vassil, 1992). 6.2.2.2 Chuyn gen bng k thut xung in K thut xung in (electroporation) l mt phng php c hc c s dng a cc phn t phn cc vo trong t bo ch qua mng t bo. Trong phng php ny, mt xung in cao th trong khonh khc (vi phn nghn giy) c kh nng lm ri lon cu trc mng kp phospholipid, to ra cc l thng tm thi cho php cc phn t DNA ngoi lai t mi trng xm nhp vo bn trong t bo. Nhiu k thut nghin cu trong sinh hc phn t yu cu a gen hoc protein ngoi lai vo trong t bo ch. V lp phospholipids kp ca mng sinh cht c

mt u a nc pha ngoi v mt u a nc pha trong (Hnh 2.14), nn bt k phn t phn cc no, bao gm c DNA v protein, u khng c kh nng i qua mng mt cch t do (Farabee, 2001).

Hnh 2.14: S mng phospholipid kp S ny cho thy cc thnh phn ha hc ca mng sinh cht. Cc u a nc phn cc hng v pha ngoi trong khi cc ui k nc hng v pha trong v tng tc vi ui k nc khc cng bm gi mng. Cc phn t phn cc khng th i qua mng ny nu nh khng c s h tr bn ngoi.

Nhiu phng php c pht trin vt qua ro cn ny, cho php a DNA v cc phn t khc vo trong t bo c nghin cu. Mt trong nhng phng php ny l k thut xung in. K thut xung in da trn trng thi tng i yu ca cc tng tc k nc ca phospholipid kp v kh nng tp hp li mt cch t ng ca n sau khi b ri lon (Purves, 2001). V vy, mt xung in chp nhong c th gy ra ri lon cc v tr ca mng mt cch nht thi, lm cho cc phn t phn cc c th i qua, nhng sau mng c th ng kn li nhanh chng v t bo khng b nh hng g c. Cc t bo ch v DNA ngoi lai c to thnh dch huyn ph v cho vo trong mt cuvette nha c in cc (Hnh 2.15).

Hnh 2.15: Cuvette nha c in cc

to ra xung in cao th trong mt thi gian ngn ngi ta s dng mt thit b gi l my xung gen (gene pulser) (Hnh 2.16). Qu trnh c bn din ra bn trong my ny c th c trnh by bng s hnh 2.17.

Hnh 2.16: My xung gen (Gene pulser) (Hng Biorad)

Hnh 2.17: S b tr mch c bn ca my xung in S ny cho thy mch in c bn cung cp in cho k thut xung in

Khi cng tc th nht ng, t in np in vo v tch mt in p cao. Khi cng tc th hai ng, in p ny phng qua dch huyn ph t bo. Mt xung in cn thit cho k thut ny thng l khong 10.000-100.000 v/cm (thay i ty theo kch thc ca t bo) trong vi phn triu giy n mt phn ngn giy. Xung in ny lm ri lon phospholipid kp ca mng t bo v to ra cc l tm thi. Kh nng in qua mng t bo cng lc tng ln 0,5-1,0 v v vy cc phn t c np in ny i qua mng t bo thng qua cc l bng cch thc tng t nh in di (Hnh 2.18).

Hnh 2.18: S plasmid cha DNA ngoi lai i qua cc l tm thi trn mng bo cht

Li DNA i vo t bo khng th quan st thy di knh hin vi, nhng hnh v ny cho thy khi nim c bn ca s to thnh cc l trn mng m DNA c th i qua. Khi cc ion np in v cc phn t i qua cc l, mng t bo phng in v cc l ny ng li mt cch nhanh chng v phospolipid kp phc hi li cu trc c (Weaver, 1995). Lc ny cc phn t mong mun trong t bo v chng c s dng cho cc nghin cu tip theo. Phng php ny c th s dng i vi gn nh tt c cc loi t bo ca cc loi. Lc u phng php ny c s dng chuyn gen vo cc t bo ng vt c v, v sau cho c t bo thc vt dng protoplast... Vi mt s cy mt l mm quan trng (loi la ph Japonica, ng, la m) m khng th th thc hin c bng phng php chuyn gen gin tip nh Agrobacterium th ngi ta thnh cng vi phng php ny. Hiu qu bin np cao. Trong mt nghin cu E.coli, 80% s t bo nhn c DNA, ngoi lai (Miller v Nickoloff, 1995). Lng DNA ngoi lai cn thit l t hn so vi cc phng php khc (Withers, 1995). Phng php ny c th thc hin vi cc m in vivo cn nguyn vn(Weaver, 1995). on DNA ngoi lai c bin np c kch thc ln. Tuy nhin nu cc xung in c cng v chiu di khng ng th mt s l ca t bo s tr nn qu ln hoc b hng khng th ng li sau khi t bo phng in, lm cho t bo b tn thng hoc b thng (Weaver, 1995). Mt hn ch na l s vn chuyn DNA ngoi lai vo v ra khi t bo trong sut thi gian in bin np

l tng i khng c hiu. iu ny dn n kt qu l khng cn bng ion m sau s lm ri lon chc nng ca t bo v t bo cht (Weaver, 1995). K thut xung in c s dng rng ri trong nhiu lnh vc khc nhau ca sinh hc phn t v y hc. Cc ng dng ca k thut xung in bao gm: - Bin np DNA: cc gen c hiu c th c to dng trong plamid v sau plasmid ny c a vo t bo ch nghin cu cu trc v chc nng ca gen v protein. - Chuyn plasmid trc tip gia cc t bo: t bo vi khun cha plasmid c th c vi mt dng khc khng mang plasmid nhng li c c tnh mong mun khc. in p ca xung in s to ra cc l, cho php mt s plasmid i ra khi t bo v li i vo t bo khc. Sau cc t bo mong mun s c chn lc bng tnh khng thuc khng sinh hoc bng phng php tng t khc (Withers, 1995). Kiu chuyn gen ny cng c th c thc hin gia cc loi khc nhau. V vy, mt lng ln plasmid sinh trng trong cc khun lc vi khun c nhn ln mt cch nhanh chng v sau c chuyn vo cc t bo nm men bng k thut xung in nghin cu (Gunn, 1995). - Dung hp t bo kch thch: s to thnh cc l thng trn mng xy ra do xung in chp nhong to ra cho thy kch thch s dung hp t bo (Weber v Berrg, 1995). - Phn phi thuc qua da: Ch khi xung in gy ra cc l tm thi trn mng sinh cht, cc l tng t to ra mng lipid kp ca lp da cht pha ngoi cng. Cc l ny cho php thuc i qua da n cc m ch. Cc bnh nhn thch phng php ny hn phng php tim (khng cn kim tim) v c th trnh c cc vn phn hy hoc hp thu khng ng ca liu php ung thuc (oral medication) trong h tiu ha (Praustmitz, 1993). - Liu php ha in khi u ung th (cancer tumor electrochemotherapy): cc nh khoa hc ang nghin cu tim nng ca k thut xung in tng tnh hiu qu ca liu php ha hc. Khi s dng k thut xung in bin np DNA, xung in ny s ph v mng t bo ung th v lm tng lng thuc i n cc v tr. Mt s nghin cu cho rng lm gim s pht trin khi u khi p dng phng php ny cho cc t bo ung th h thng m hnh ng vt (Maeda, 1998). - Liu php gen: k thut xung in cho php cc vector mang cc gen quan tm c bin np qua da n cc m ch. Khi hp nht vo cc t bo ca c th, cc protein c tng hp t cc gen ny c th thay th gen sai hng v v vy iu tr cc ri lon di truyn (Hnh 2.19)(Inovio, 2002).

Hnh 2.19: ng dng ca k thut xung in trong liu php gen

6.2.2.3. Chuyn gen bng sng bn gen Sng bn gen (Gene gun) l mt thit b s dng a thng tin di truyn vo t bo, c thit k u tin cho bin np DNA ngoi lai vo t bo thc vt v c pht trin vo u thp nin 1980 do cc nh thc vt hc i hc Corrnell cng vi cc nh nghin cu Corrnell Nanofabrication Facility, Newyork, USA. Sng bn gen c bn trn th trng vo nm 1990. n s dng cho loi sng ny l cc ht kim loi nng c bn c bao bc DNA. Tn chnh xc v y ca sng bn gen l h thng phn phi ht biolistics (biolistic particle delivery system) v k thut ny thng c gi mt cch n gin l biolistics (s kt hp gia hai thut ng biology (sinh hc) v ballistics (s bn tung)). Mc d c nhiu thit k k thut khc nhau nhng nguyn l chung ca phng php ny l s dng p lc xung ca kh helium gai tc cc ht.

Hnh 2.20 : Sng bn gen (Hng Biorad)

Sng bn gen bao gm hai bung bng thp khng g, kch thc 6x7x10 ni vi hai bm chn khng. DNA ngoi lai c gn vo cc ht tungsten c ng knh rt nh, khong 1m (cc kim loi nng khc nh vng v bc cng c s dng nhng khng thng xuyn do gi c t). Cc ht ny c t trn mt

ci a mt bn trong ca sng. S bng n kh helium 1000psi lm cho ci a bn v pha trc vi tc 1300 food/s, tng ng vi tc khi mt vin n ri khi nng sng. Mt tm chn lm dng a li v cc ht vng hay tungsten c phng v pha cc t bo ch. Chng xuyn qua vch t bo v phng thch cc phn t DNA (Hnh 2.21). Sng bn gen s dng k thut DNA ti t hp hp nht s biu hin cc gen phn phi. Cc t bo bin i di truyn c th c s dng to thc vt bao gm c s sa i di truyn mong mun trong tt c cc t bo ca chng (Voiland, 1999).

Hnh 2.21: S nguyn l hot ng ca sng bn gen

Mc tiu ca sng bn gen thng l callus ca cc t bo thc vt ging nhau sinh trng trong mi trng gel trn a petri. Sau khi cc ht tungsten va chm vo a, gel v callus b ph v nhiu. Tuy nhin mt s t bo khng b ph v khi va chm mnh v tip nhn cc ht tungsten c bao bc DNA v cui

cng cc phn t DNA ngoi lai xm nhp v hp nht vo nhim sc th thc vt. Cc t bo t a petri c tp hp li v chn lc cc t bo hp nht thnh cng v biu hin DNA ngoi lai bng cc k thut ha sinh hin i nh s dng gen chn lc ni tip v Northern blots. Cc t bo n chn lc t callus c th c x l vi mt s hormone thc vt nh auxin, gibberelin v mi mt t bo c th phn chia, bit ha thnh cc t bo m, c quan, t bo chuyn ha ca ton b cy. Cy mi c ngun gc t mt t bo ny mm thnh cng c th mang cc c tnh di truyn mi.

Hnh 2.22: Chuyn gen bng sng bn gen Phng php ny c u im l thao tc d dng, c thchuyn gen vo nhiu loi t bo v m, cc t bo c bin np c t l sng st cao, cho php a cc gen vo t bo v tr mong mun....Do vy n c s dng rng ri trong nhiu lnh vc. Cc ng dng ca k thut bn gen bao gm - To thc vt chuyn gen: y l phng php c s dng rng ri nht hin nay trong lnh vc to ng cc chuyn gen. Bacillus thuringiensis l loi vi khun tng hp ra protein crystal (crys1Ab v crys1Ac) c kh nng git mt cch chn lc cc nhm cn trng nht nh. Gen m ho protein crystal c gi l ge Bt. Gen Bt c bn vo m so ca cy ng cc (Rassmussen, 1994). Trong khi cc t bo ny sa cha tn thng, DNA ngoi lai xm nhp vo genome t bo ch. V vy cho php t bo ch phin m v gii m gen Bt. Sau mi ln qu trnh bin np c hon thnh ngi ta cng tin hnh sng lc theo phng php truyn thng l da trn c s cc marker chn lc c xen vo DNA cu trc (Brettschneider, 1997). Cc marker chn lc mang tnh khng (khng thuc khng sinh hay khng thuc dit c) nh kanamycin l mt l mt trong nhng marker ph bin nht c s dng.

- Tim chng vaccine di truyn: cc gen c a vo c th bng sng bn gen vi mc ch gy ra phn ng min dch vi protein biu hin bi gen chuyn. Phng php tim chng vaccine ny an ton hn cc phng php khc bi v ch DNA ngoi lai c a vo v khng c cc protein ngoi lai (Lin, 2000). - Liu php gen t st (Suicide gene therapy): phng php bn gen c s dng trong iu tr bnh ung th. Mt gen biu hin protein gy c c promoter c hiu khi u c a vo cc t bo khi u. Khi protein ny c biu hin th t bo khi u cht. Protein ny ch gy c i vi cc t bo khi u bi v promoter c hiu cn cho s biu hin ch c to ra trong cc t bo khi u (Lin, 2000). - S iu bin min dch (Immunomodulation): phng php ny cn c s dng chng li ung th. S dng sng bn gen, mt protein ch biu hin trong t bo khi u nhng lm cho phn ng min dch tng ln c a vo t bo. Phn ng min dch tng ln nhm vo cc t bo khi u v hin nhin gy ra hiu qu mong mun (Lin,2000). - Dc l di truyn: sng bn gen c th c s dng a cc gen tng hp protein hu ch hay protein liu php vo c th. V d nh cc yu t ng mu cc c th ri lon s ng mu hoc tng s tng hp hng cu trong cc c th thiu mu. S biu hin ko di ca cc gen a vo l mt vn , trong nhiu trng hp thng i hi s phn phi gen phc tp (Lin, 2000). - Sng bn gen l mt cng c nghin cu: sng bn gen c th c s dng xen cc promoter m s dn n s biu hin ca cc gen nht nh. Hiu qu khuych i cc protein nht nh l mt phng php c gi tr ln i vi cc nh khoa hc nghin cu chc nng ca cc protein ny (Lin, 2000).

Hnh 2.22: Chuyn gen bng sng bn gen


6.2.2.5.K thut chuyn gen bng sc nhit

Dng nhit to ra nhng bin i 6.2.2.6. K thut chuyn gen qua ng phn (polen tuber)

Phng php ny cn c xem l phng php chuyn khng qua nui cy m. Chuyn gen trc tip thng qua ng phn c nhm Ray Wu i hc Cornell (M) bo co thnh cng trn cy la, tip theo cc cng trnh s b vi ADN tng hp ca hai tc gi Trung Quc Duan v Chen (1985). y l phng php li dng ng phn chuyn vector mang gen i cng t bo sinh dc c( tinh t) kt hp vi t bo trng to hp t mang gen ngoi lai c chuyn vo. Sau , hp t s pht trin thnh ht. Ht ny mm v pht trin thnh cy chuyn gen v c di truyn cho cc th h sau. Chuyn gen qua ng phn tt nht thc hin ngay sau khi qu trnh th tinh xy ra non, nhng t bo sinh dc ci cha kp phn chia. Nu lm c nh vy s chuyn gen s c thc hin i vi mt t bo sinh sn ci (trng) duy nht v sau khi ti sinh cy s trnh xut hin th khm. Trong th nghim ca Ray Wu, t l ht chuyn gen/ht th nghim l 20%.

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