chuyen gen vao co the dong va thuc vat

8/3/2019 Chuyen Gen Vao Co the Dong Va Thuc Vat

1/31

1. Chuyn genS chuyn gen thc vt dng A. tumefaciens da trn nguyn l nh hnh sau:

Hnh: Agrobacterium mang plasmid gy khi u thc vt (tumour inducing plasmid- Tiplasmid), plasmid ny cha cc gen vir v vng gen cn chuyn (T-DNA). Gen quan tm cchn vo vng T-DNA. Cc t bo tn thng thc vt tit ra hp cht bo v, hp cht nykch thch s biu hin gen vir Agrobacterium . Vir protein to ra T-strand t vng T-DNAtrn Ti-plasmid. Sau vi khun gn vo t bo thc vt, T-strand v mt s protein virchuyn vo t bo thc vt qua knh vn chuyn. Trong t bo thc vt, cc protein vir tngtc 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 nm2005), Broothaerts v cng s tm ra cch chuyn gen thc vt dng 3 loi vikhun mi (1). Trong cng trnh ca h, tc gi dng 3 chng Rhizobium loiNGR234, Sinorhizobium meliloti v Mesorhizobium loti th nghim chuyn gen vomt s loi thc vt khc nhau thuc hai nhm thc vt bc cao: nhm mt l mmv nhm hai l mm. C ch chuyn gen ca 3 loi vi khun ny v thc vt gingvi c ch chuyn gen ca Agrobacterium . Hiu qu chuyn gen ca 3 vi khun nytng i thp, t 1 n 40% so vi Agrobacterium . Tuy nhin tc gi cho rng nuthay i mt s nhn t nh tui v loi m thc vt c th nng cao hiu quchuyn gen. Tn sut chuyn gen cao ca Agrobacterium l kt qu ca qu trnhnghin 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 haca thc vt.

Dng A. tumefaciens chuyn gen cc nh khoa hc gp mt s kh khn v lutbn quyn v iu ny lm cho gi thnh tng cao gy kh khn cho nghin cucng 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 CAMBIAcpbng sng ch. CAMBIA l mt t chc phi li nhun s hu nhiu sng ch theo
http://www.cambia.org/http://www.cambia.org/


2/31

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

Chuyn gen (transgenesis) l a mt on DNA ngoi lai vo genome ca mt cth a bo, sau on DNA ngoi lai ny s c mt hu ht cc t bo v truyn li cho th h sau. V vy khi nim chuyn gen ch c s dng cho thvt v ng vt. Nm men, vi khuNn v t bo nui cy mang mt on DNngoi lai c gi l cc t bo ti t hp (recombinant cell) hoc t bo bin n(transformed cell).Chuyn gen khc vi liu php gen (gene therapy). C trng hp cc t bo mkhng mang DNA ngoi lai. Thut ng liu php gen mm (germinal genetherapy) cng c s dng. Liu php gen mm hy cn cha c th nghimngi. Cc t bo mm ny mang DNA ngoi lai v c truyn li cho th hsau.V mt lch s, thut ng GMO (genetically modified organism)-sinh vt bin

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 chnxc hn, GMO cp ti tt c cc c th sng bin i di truyn, bao gm csinh vt. Thut ng GMP (genetically modified plant)-thc vt bin i gen vGMA (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 ghu 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), rybozym

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 sinvt chuyn gen. DNA ngoi lai khng th tn ti trong c th m khng hp nhvo trong genome ca n. Mt on DNA t do nhanh chng b loi tr trong chtrnh t bo v vy n s khng c kh nng ti bn v truyn li cho cc t bcon. Tuy nhin v l thuyt th c th duy tr mt on DNA ngoi lai nh mnhim sc th nh (minichromosome) c kh nng t ti bn v c mt trong ct bo con. Mt s genome virus c c tnh ny, v d nh virus herpes. Mt von nhim sc th thng c tm thy cc t bo khi u, l cc nhim sctn ti trong mt thi gian ngn, mang cc yu t ti bn v truyn cho cc t bcon.2. ng vt (Thc vt) chuyn genng vt (Thc vt) chuyn gen l ng vt (thc vt) c gen ngoi lai (gechuyn) 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 ssn mm. Nu dng t bo mm b bin i, cc tnh trng b bin i ny s truyn cho cc th h k tip thng qua qu trnh sinh sn bnh thng. Nu ch dng t bo sinh dng b bin i, ch c c th mang cc t bo sinh dng


3/31

b nh hng v khng di truyn li cho th h sau. Vic chuyn gen ngoi lai vng 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 vng vt chuyn gen. ng vt, khng ch i vi ng vt m hnh (chut), vnui (b, ln, d, cu, th, g, c...) m c nhng loi ng vt khc nh kh, muv mt s cn trng...3. Gen chuynGen chuyn (transgene) l gen ngoi lai c chuyn t mt c th sang mt cth mi bng k thut di truyn.Cc gen chuyn c s dng to ng vt, thc vt chuyn gen c ngun t cc loi sinh vt khc nhau: ng vt, thc vt, vi sinh vt v c con ngi. Vd: gen ca ngi c a vo chut v cc vt nui khc nh ln, b, cuchim...4.Mc ch ca vic chuyn gen Ni chung, mc ch ca chuyn gen l thm mt thng tin di truyn ngoi lai vgenome, cng nh c ch mt gen ni sinh. Trong mt s trng hp, s thth mt gen hot ng chc nng bng mt gen hot ng chc nng khc l cthit. Gen ngoi lai c th l mt th t bin ca gen ni sinh hoc mt gen hoton khc.S thm gen c th c thc hin cung cp sinh vt mang protein mi. Sthm gen cng c th c s dng nghin cu c ch hot ng ca m promoter trong ton c th. S kt hp ca gen reporter vi promoter l nguytc chung ca phng php ny.S thay th gen c s dng ch yu lm bt hot mt gen bit. Trn tht, n bao gm s thay th gen ni sinh bng mt th t bin bt hot. Ph php ny c dng cho thng tin v chc nng sinh hc ca gen nh trhp thm gen. Thc vy c thm gen v bt hot gen c th gy ra cc bin isinh vt chuyn gen, m cc bin i ny c th c quan st hoc nh gi. Cth t bin ca gen thay th c th cung cp thng tin bng mt cch thc tinhhn.S thay th mt gen bng mt gen c chc nng khc nhau hon ton l kh hnhng c th thc hin a mt marker hoc mt gen chn lc vo genome. 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 hvi gen ngoi lai vo ng vt (thc vt) (do con ngi ch ng to ra). Cc gngoi lai ny phi c truyn thng qua dng mm v vy mi t bo k cc bo mm sinh sn ca ng vt (thc vt) u cha vt cht di truyn ci nh nhau.6.Cc phng php chuyn gen


4/31

Nhiu phng php khc nhau c pht minh a gen vo t bo v mng vt v thc vt. K thut n gin nht l chuyn DNA trn bng vi ti(microinjection), xung in(electroporation), sng bn gen. Cc phng phpvector virus, t bo gc phi, chuyn gen gin tip nh vi khun phc tp v hiqu 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 ngila chn phng php chuyn gen ph hp.6.1.T bo ng vt 6.1.1. DEAE-dextranDEAE-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 sc mt ca DEAE-dextran. DEAE-dextran s kt hp vi DNA tch in m (Hn2.1). S d tha in tch dng trong phc hp DNA-polycation do s ng gca polycation, cho php phc hp ny i n kt hp cht ch hn vi mng bo tch in m. S xm nhp ca phc hp c th t c nh s nhp b(endocytosis).

Hnh 2.1: S kt hp gia DEAE-dextran v DNAPhng php ny c s dng thnh cng trong vic chuyn DNA vo cc t b biu hin nht thi, c ngha l thi gian biu hin ngn ch vi ngy trong qutrnh nghin cu. Tuy nhin, phng php ny ni chung l khng hu ch i v

cc nghin cu cn s chuyn nhim n nh da vo s hp nht ca DNA nglai vo nhim sc th, hiu qu bin np thp, ch c s dng cho mt s lo bo v cho kt qu tt i vi t boex 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


5/31


6/31

np khng ng k, s biu hin gen c th l nht thi hoc n nh v qutrng trong vic thit k vector virus ti t hp. Tuy nhin hiu qu bin np mc biu hin ca gen chuyn thp.6.1.3. Phng php vi timS thnh cng u tin trong nghin cu to chut chuyn gen bng phng phvi tim vo tin nhn c cng b vo nm 1980 (Gordon, 1980). Mc ducu trc t hp gen chuyn c chng minh l tch hp vo genome cchut, n c sp xp li nhng gen ngoi lai khng c biu hin. Cc c 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. Vnm 1982, ln u tin s thay i kiu hnh c th nhn thy chut nht chuygen c m t (Palmiter, 1982). y l kt qu biu hin ca gen hormon sintrng chut cng chut nht. T n nay c rt nhiu cc cng trnh chuyn gen, trong phn ln l cc nghin cu hiu qu ca gen vi tim vi ssinh trng ca ng vt c v v bnh hc.

Nguyn tc ca phng php vi tim l mt lng nh DNA c tim trc tivo nhn t bo phi trn hoc t bo nguyn vn mt cch c hc di knh hivi. Phng php ny cho php a gen vo ng v tr mong mun tng t bovi hiu qu tng i cao.Tuy nhin do i hi phi tinh vi, t m v cc k chnxc nn hn ch s lng t bo vi tim v ngoi ra cn c th lm tn thng 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 ktim v kim gi. Kim c to ra t nhng ng thu tinh do capillar ng kn0,1-1,5 mm c si bng wolfram mnh trong nh h thng thit b lm kim. Hthng ny gm c my ko kim t ng (pipette puller), my mi kim v my gia

c kim.


7/31

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 dng my gia c ct u nhn ca ng capillar sau khi ko to ra mi kim tic ng knh thch hp. ng knh bn trong mi kim tim tu thuc vo lot bo chuyn gen. i vi trng tin nhn ng vt c v c ng knh kho70 m th ng knh ca kim tim khong 0,75 m l thch hp, i vi phi cmt t bo ng knh ca kim tim l khong 3-4 m. Cui cng a kim lmy mi lm sc nhn v trn lng u kim (Hnh 2.8). Kim gi c ch tt nhng ng capillar c u nhn bnh thng c nh trng trong qu trnh 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 kvi ng knh thch hp. Lm nhn u kim gi bng cch ng kim ny ngtrn mt on cong platin ca my gia c. t nng si platin v iu chnh 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 gc hai b phn chnh l knh hin vi v my vi thao tc.


8/31

Hnh 2.8 : Cc my lm kim (Hng Narishige)1. My gia c kim Microforge2. My mi kim3. My ko kim t ng Pipette Puller Knh hin vi dng cho mc ch ny l knh hin vi soi ngc (vt knh xoayngc 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, mdng iu chnh kim tim, mt dng cho kim gi. Tnh nng ca my ny lcho php iu chnh cc kim theo khng gian 3 chiu. Kim tim v kim gi clp vo my vi thao tc v c ni vi syringe qua ng bng cht do c ny du parafin.Chun b dung dch gen chuyn: gen chuyn c xen vo tronmt vector plasmid v to dng trong E.coli . Cc vi khun bin np mang plasmidti 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 sintrng trong mi trng dinh dng thch hp v plasmid ti t hp mang genchuyn s c sao chp mi khi t bo vi khun phn chia. Sau , hng tri bn sao ca plasmid ti t hp mang gen chuyn c tch chit t cc t bo khun ny v cc on gen chuyn c tch ra t plasmid ti t hp nh s d

enzym hn ch. tinh sch, gen chuyn c in di trn gel agarose. Ho tagen chuyn trong dung dch m c trng (nh dung dch Tris-EDTA; dung dchTE...) v tnh nng dung dch gen chuyn nh quang ph k. Nng dundch DNA s dng cho vi tim thng l 1-5 g/ml. Trc khi chuyn vo phigen chuyn c kim tra s biu hin trong cc t bo nui cy bng s chuynhim (transfection).


9/31

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 phcapillar (ngm u kim tim vo dung dch gen khong 10-12 gi) hoc bm trtip dung dch gen vo, lp kim tim v kim gi vo my vi thao tc, chuyn trn

tin nhn vo a petri c cha mi trng c t di knh hin vi, gi trntin nhn vo u kim gi bng lc ht ca syringe, iu chnh knh hin vi xnh a phi v iu chnh my vi thao tc a kim tim vo v tr ca trngtin nhn, y gen vo trng tin nhn bng cch vn nh syringe. Khi thy trtin nhn hi phng to v tr nn sng hn th dng li v ko nhanh kim tim raTrng tin nhn sau khi tim c di chuyn xa n cui a petri trc khi timtrng tin nhn tip theo. Mi mt nhm trng tin nhn hon thnh chuyn sang mt a mi trng khc p v nh gi bng mt trong mt vting. Sau tt c cc trng tin nhn c nhn thy r rng v c chuyn vng dn trng ca con ci nhn.

Cho n nay, trong cc k thut chuyn gen vo ng vt th phng php vi tidung dch DNA vo hp t l phng php c hiu qu nht trn ng vt c vv 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 phca 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 trnngu nhin v xc sut gen chuyn xen vo v tr DNA vt ch m s cho phn biu hin l thp. Hiu qu ca vi tim l khng cao (Bng 2.1).


10/31

Bng 2.1: Hiu qu vi tim mt s loi ng vt

Loing vt

S lngtrng cvi tim

S lngcon sinh ra t trng vi tim

S lngcon chuyngen

Th 1907 218 (11,4%) 28 (1,5%)Cu 1032 73 (7,1%) 1(0,1%)Ln 2035 192 (9,4%) 20 (1%)


11/31

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


12/31

hp vi DNA tch in m v kt qu l cha y DNA trong phc hp liposomDNA (Hnh 2.5). i vi cc t bo nui cy, ton b li tch in dng c phc hp liposome-DNA ni chung l gy ra hiu qu chuyn gen cao hn bi vn cho php phc hp ny kt hp vi mng t bo tch in m bn hn. Nh 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 nhnnh th no. DOPE c xem l mt lipid kch thch s dung hp v vai tr cn l phng thch cc phc hp ny t endosome cng nh lm cho s dung hpca 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 t phospholipid. Cc loi ti khc nhau c m t, nhng ti mt lp mng lthch hp nht cho chuyn gen v chng c t l khong trng cha nc btrong tng i cao i vi mi n v lipid v bi v chng c t l 59 phn phcao hn. S dung hp ca liposome vi mng plasma l mt s kin him. Hiqu bin np ca phng php ny thp hn so vi phng php vi tim vo ti

nhn. Cc n lc nghin cu ang c tin hnh tm ra cc iu kin thnghim m c th lm tng s phng thch cc phn t kt nang t con nm bo.

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

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


13/31

Hnh 2.5: Phc hp liposome-DNA

Hnh 2.6: S hot ng ca vector liposome


14/31

Liposome c s dng a protein, lipid v cc phn t nh vo nhiu lot bo nui cy, tuy nhin hiu qu thp hn vi tim i vi RNA hoc proteiCng nh th, chuyn gen qua liposome v s biu hin ca gen chuyn l khnvt qua c cc phng php chuyn gen thng thng (nh h thng virus), biu hin gen chuyn thng nht thi, s c ch bi cc thnh phn ca huthanh c th xy ra. Bn cnh , k thut ny c nhiu u im l gen chuynkhng hp nht vo genome ch, c hiu qu tt i vi c t boin vitro v invivo, c th mang c cc DNA c kch thc rt ln, tinh khit cao, khngy min dch, c th s dng vi cc t bo m bin np bng k thut calci phosphate khng c hiu qu..ng dng trong tng lai ca k thut ny ch yu s l kh nng phn phi thuvo cc t bo ca c th sng. Hin nay k tht ny ang c pht trin tin s phn phi c hiu ca liposome bng cch ghp cc khng th c hivi 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 timDNA ca retrovirus SV40 vo khoang phi (blastocoel) ca ti phi chut, DNAny 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 trongcc th nghim nh th ny tch hp vo genome v truyn li cho th h sado 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 trinPhng php ny tuy thao tc hi phc tp nhng c u im l hiu qu chuygen cao. Hn na gen cu trc gn vo vector virus s s dng promoter ca virucc promoter ny thng c tnh cao do gen cu trc ny s c biu hi

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 , c 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 bgiai on sm ca phi trc khi c chuyn ghp vo con m. Gen chuyn vvector retrovirus xm nhp mt cch hiu qu vo h gen ca vt ch nhng virs 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 lkhng phi tt c cc t bo ca c th u mang retrovirus. Gen chuyn ch c tdi truyn c nu retrovirus hp nht vo mt s t bo sinh dc. i vi ph php ny t l sng ca cc ng vt chuyn gen s sinh l rt thp. Nu nh cthao tc di truyn l chun xc, khng gy ra s sy thai, th th h ng vt tin (F1) cn kim tra s biu hin ca gen chuyn. Khi gen chuyn hp nhtrong cc t bo sinh dc th c gi l th khm dng mm v sau chnc lai cng dng khong 10-20 th h cho n khi thu c cc ng v


15/31

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

Hnh 2.11: Chuyn gen nh vector l virus6.1.6. Chuyn gen bng cch s dng t bo gc phiXut pht t l do cc t bo gc phi (t bo phi giai on 16-32 t bo) l ct 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 tihnh tch chit cc t bo gc phi v bin np gen ngoi lai vo nhng t bny.Sau khi chn ra nhng t bo c bin np gen l ngi ta a n vo phkhc giai on phi nang to ra ng vt chuyn gen th khm. Trn 30


16/31

ng vt chuyn gen to thnh l nhng ng vt chuyn gen th khm dng mmang kiu gen ca dng t bo ny. y cc t bo gc phi c s dng nhl mt phng tin chuyn gen (Mintz, 1977).u im ca phng php chuyn gen ny l t l phi sng st sau thao tc, stch hp v biu hin tnh trng ca gen mi kh cao. iu quan trng hn l tronthc t vic chuyn gen c th c tin hnh thng qua s thao tc vi phi dv ti phi. Phi cc giai on ny c th thu nhn m khng cn phu thu(c bit l i vi b), do vy cng vic chuyn gen c tin hnh rt d dnPhng php ny c ngha c bit i vi s nghin cu kim tra di truyn ccc qu trnh pht trin. S thun li ca n l cho php to ra mt cch chnh xcc 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 rito 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 ph

(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.


17/31

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


18/31

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 dng Agrobaterium. Pht kin v vic Agrobaterium tumefaciensc kh nngchuyn gene vo thc vt bin loi ny tr thnh mt trong nhng cng cquan trong nht ca Cng ngh sinh hc thc vt.6.2.1.Phng php gin tip6.2.1.1. Phng php chuyn gen gin tip nh Agrobacterium

A. tumefaciens l loi vi khun t c kh nng chn gene ca mnh vo thc vtv sau s dng b my thc vt biu hin cc gene ny di dng hp cdinh dng cho chng. A. tumefaciens s to ra cc khi u thc vt gn im tipni gia r v thn, gy ra nhng nt sn cy (bnh mn cy-crown gal

disease). Agrobacterium c kh nng xm nhim t bo thc vt bng cch chuyn mton DNA ca n vo t bo thc vt. Khi DNA vi khun c hp nht vnhim sc th thc vt, n s tn cng vo h thng t chc ca t bo mt cc hiu qu v s dng n m bo cho s sinh si ca qun th vi khun. Tkhng may mn cho cc nh trng cy n qu khi gp phi loi vi khun ny. Bv n chnh l th phm gy ra bnh khi u hnh chp v bnh lng r nhiu locy cnh v cy n qu.


19/31

Mc d h thng chuyn gen gin tip nh Agrobacterium l c hiu qu i vimt s loi nhng khng phi tt c thc vt c th c bin np bng con ny. c bit, lp mt l mm bao gm cc cy ng cc chnh trn th gii nhla, 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 nhkhoa hc loi b cc gen gy khi u v gen m ho opine ca T - DNA v thayth vo l cc marker chn lc, trong khi vn duy tr cc vng b phi v btri ca T-DNA v cc gen vir. Gen chuyn c xen vo gia cc vng b caTDNA. N s c chuyn vo t bo v tr nn hp nht vi nhim sc th bo thc vtPhng php chuyn gen gin tip nh Agrobacterium c kim tra i vis xm nhp bn vng, s biu hin v s di truyn ca cc gen chuyn c bTuy nhin, mt vi yu t nh hng n hiu qu bin np l loi m c bnp, giai on pht trin ca m, mc khi u ca vi khun A. tumefaciens sdng, mi trng nui cy m sau khi bin np, marker c s dng ch

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 tip6.2.2.1. Chuyn gen trc tip vo protoplast DNA d xm nhp c vo t bo thc vt, phi loi b vch t bo t protoplast. Protoplast c th c duy tr trong mi trng nui cy nh cc t bsinh trng mt cch c lp hoc vi mt mi trng c hiu, vch t bo c c to thnh v ton b cc cy c th c ti sinh t cc t bo ny. Qu trnchuyn gen nh th ny c thc hin mt cch trc tip bng mt c ch v

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 chiu qu, c bit i vi nhng loi thc vt m phng php chuyn gen gitip nh Agrobacterium khng th thc hin c. Tuy nhin, vic to protoplastcng nh ti sinh cy t protoplast khng n gin, tn nhiu cng sc, b nhng 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 phn php ny, mt xung in cao th trong khonh khc (vi phn nghn giy) c khnng lm ri lon cu trc mng kp phospholipid, to ra cc l thng tm thcho php cc phn t DNA ngoi lai t mi trng xm nhp vo bntrong t bo. Nhiu k thut nghin cu trong sinh hc phn t yu cu a gen hoc protengoi lai vo trong t bo ch. V lp phospholipids kp ca mng sinh cht c


20/31

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

Hnh 2.14: S mng phospholipid kpS ny cho thy cc thnh phn ha hc ca mng sinh cht. Cc u a nc phcc hng v pha ngoi trong khi cc ui k nc hng v pha trong v tng tvi ui k nc khc cng bm gi mng. Cc phn t phn cc khng th i qumng ny nu nh khng c s h tr bn ngoi. Nhiu phng php c pht trin vt qua ro cn ny, cho php DNA v cc phn t khc vo trong t bo c nghin cu. Mt trong nhn phng php ny l k thut xung in.

K thut xung in da trn trng thi tng i yu ca cc tng tc k nca phospholipid kp v kh nng tp hp li mt cch t ng ca n sau khi bri lon (Purves, 2001). V vy, mt xung in chp nhong c th gy ra ri lo cc v tr ca mng mt cch nht thi, lm cho cc phn t phn cc c th qua, nhng sau mng c th ng kn li nhanh chng v t bo khng b nhhng g c.Cc t bo ch v DNA ngoi lai c to thnh dch huyn ph v cho vo tronmt cuvette nha c in cc (Hnh 2.15).


21/31

Hnh 2.15: Cuvette nha c in cc to ra xung in cao th trong mt thi gian ngn ngi ta s dng mt thigi 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 hn2.17.

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


22/31

Hnh 2.17: S b tr mch c bn ca my xung inS ny cho thy mch in c bn cung cp in cho k thut xung inKhi cng tc th nht ng, t in np in vo v tch mt in p cao. Kcng tc th hai ng, in p ny phng qua dch huyn ph t bo. Mt xunin cn thit cho k thut ny thng l khong 10.000-100.000 v/cm (thay ty theo kch thc ca t bo) trong vi phn triu giy n mt phn ngn giXung in ny lm ri lon phospholipid kp ca mng t bo v to ra cc l tthi. Kh nng in qua mng t bo cng lc tng ln 0,5-1,0 v v vy cc phnt c np in ny i qua mng t bo thng qua cc l bng cch thc tt nh in di (Hnh 2.18).


23/31

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

Li DNA i vo t bo khng th quan st thy di knh hin vi, nhng hnh vny cho thy khi nim c bn ca s to thnh cc l trn mng m DNA c thi qua.

Khi cc ion np in v cc phn t i qua cc l, mng t bo phng in vcc l ny ng li mt cch nhanh chng v phospolipid kp phc hi li cu trc (Weaver, 1995). Lc ny cc phn t mong mun trong t bo v chngc 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 loi. Lc u phng php ny c s dng chuyn gen vo cc t bo vt c v, v sau cho c t bo thc vt dng protoplast... Vi mt s cy mmm quan trng (loi la ph Japonica , ng, la m) m khng th th thc hinc 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 (Withers1995). Phng php ny c th thc hin vi cc min vivo cn nguynvn(Weaver, 1995). on DNA ngoi lai c bin np c kch thc ln. Tunhin nu cc xung in c cng v chiu di khng ng th mt s l ca bo s tr nn qu ln hoc b hng khng th ng li sau khi t bo phng ilm cho t bo b tn thng hoc b thng (Weaver, 1995). Mt hn ch na l vn chuyn DNA ngoi lai vo v ra khi t bo trong sut thi gian in bin n


24/31

l tng i khng c hiu. iu ny dn n kt qu l khng cn bng ion 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 sihc 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 gv protein.- Chuyn plasmid trc tip gia cc t bo: t bo vi khun cha plasmid c tc vi mt dng khc khng mang plasmid nhng li c c tnh mong mukhc. in p ca xung in s to ra cc l, cho php mt s plasmid i ra khi bo v li i vo t bo khc. Sau cc t bo mong mun s c chn lc btnh khng thuc khng sinh hoc bng phng php tng t khc (Withers1995). Kiu chuyn gen ny cng c th c thc hin gia cc loi khc nhauV vy, mt lng ln plasmid sinh trng trong cc khun lc vi khun nhn ln mt cch nhanh chng v sau c chuyn vo cc t bo nm men

bng k thut xungin nghin cu (Gunn, 1995).- Dung hp t bo kch thch: s to thnh cc l thng trn mng xy ra dxung in chp nhong to ra cho thy kch thch s dung hp t bo (Webev Berrg, 1995).- Phn phi thuc qua da: Ch khi xung in gy ra cc l tm thi trn mng sinhcht, cc l tng t to ra mng lipid kp ca lp da cht pha ngoi cnCc 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 trnhc cc vn phn hy hoc hp thu khng ng ca liu php ung thuc (o

medication) trong h tiu ha (Praustmitz, 1993).- Liu php ha in khi u ung th (cancer tumor electrochemotherapy): cc nhkhoa hc ang nghin cu tim nng ca k thut xung in tng tnh hiu qca liu php ha hc. Khi s dng k thut xung in bin np DNA, xuin ny s ph v mng t bo ung th v lm tng lng thuc i n cc v tMt s nghin cu cho rng lm gim s pht trin khi u khi p dng phn 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 tmc bin np qua da n cc m ch. Khi hp nht vo cc t bo ca c thcc 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).


25/31

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


26/31

6.2.2.3. Chuyn gen bng sng bn genSng bn gen (Gene gun) l mt thit b s dng a thng tin di truyn vo bo, c thit k u tin cho bin np DNA ngoi lai vo t bo thc vtc pht trin vo u thp nin 1980 do cc nh thc vt hc i hc Corrncng 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 snny 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 deliverysystem) v k thut ny thng c gi mt cch n gin l biolistics (s khp gia hai thut ng biology (sinh hc) v ballistics (s bn tung)). Mc d cnhiu thit k k thut khc nhau nhng nguyn l chung ca phng php ny 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 nvi hai bm chn khng. DNA ngoi lai c gn vo cc ht tungsten c nknh rt nh, khong 1m (cc kim loi nng khc nh vng v bc cng c sdng nhng khng thng xuyn do gi c t). Cc ht ny c t trn mt


27/31

ci a mt bn trong ca sng. S bng n kh helium 1000psi lm cho ci bn v pha trc vi tc 1300 food/s, tng ng vi tc khi mt vin ri khi nng sng. Mt tm chn lm dng a li v cc ht vng hay tungstec phng v pha cc t bo ch. Chng xuyn qua vch t bo v phng thchcc 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 tc s dng to thc vt bao gm c s sa i di truyn mong mun trtt c cc t bo ca chng (Voiland, 1999).

Hnh 2.21: S nguyn l hot ng ca sng bn genMc tiu ca sng bn gen thng l callus ca cc t bo thc vt ging nhasinh trng trong mi trng gel trn a petri. Sau khi cc ht tungsten vachm vo a, gel v callus b ph v nhiu. Tuy nhin mt s t bo khng b phv khi va chm mnh v tip nhn cc ht tungsten c bao bc DNA v cu


28/31

cng cc phn t DNA ngoi lai xm nhp v hp nht vo nhim sc th thvt. Cc t bo t a petri c tp hp li v chn lc cc t bo hp nhthnh cng v biu hin DNA ngoi lai bng cc k thut ha sinh hin i nh 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 thvt nh auxin, gibberelin v mi mt t bo c th phn chia, bit ha thnh cc bo m, c quan, t bo chuyn ha ca ton b cy. Cy mi c ngun gc tmt t bo ny mm thnh cng c th mang cc c tnh di truyn mi.

Hnh 2.22: Chuyn gen bng sng bn genPhng php ny c u im l thao tc d dng, c thchuyn gen vo nhiu lot bo v m, cc t bo c bin np c t l sng st cao, cho php a cc gevo t bo v tr mong mun....Do vy n c s dng rng ri trong nhiu lnvc. 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 hinay trong lnh vc to ng cc chuyn gen. Bacillus thuringiensis l loi vi khuntng hp ra protein crystal (crys1Ab v crys1Ac) c kh nng git mt cch chnlc 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 khicc t bo ny sa cha tn thng, DNA ngoi lai xm nhp vo genome t bch. 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 phtruyn thng l da trn c s cc marker chn lc c xen vo DNA cu tr(Brettschneider, 1997). Cc marker chn lc mang tnh khng (khng thuc khngsinh hay khng thuc dit c) nh kanamycin l mt l mt trong nhng marke ph bin nht c s dng.


29/31

- Tim chng vaccine di truyn: cc gen c a vo c th bng sng bn gevi mc ch gy ra phn ng min dch vi protein biu hin bi gen chuyPhng php tim chng vaccine ny an ton hn cc phng php khc bi vch 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 sdng trong iu tr bnh ung th. Mt gen biu hin protein gy c c promotc hiu khi u c a vo cc t bo khi u. Khi protein ny c biu hint 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 kh(Lin, 2000).- S iu bin min dch (Immunomodulation): phng php ny cn c sdng chng li ung th. S dng sng bn gen, mt protein ch biu hin trot bo khi u nhng lm cho phn ng min dch tng ln c a vo t bPhn ng min dch tng ln nhm vo cc t bo khi u v hin nhin gy ra hiqu mong mun (Lin,2000).

- Dc l di truyn: sng bn gen c th c s dng a cc gen tng h protein hu ch hay protein liu php vo c th. V d nh cc yu t ng mucc c th ri lon s ng mu hoc tng s tng hp hng cu trong cc c tthiu mu. S biu hin ko di ca cc gen a vo l mt vn , trong nhitrng 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 qkhuych i cc protein nht nh l mt phng php c gi tr ln i vi cnh khoa hc nghin cu chc nng ca cc protein ny (Lin, 2000).


30/31

Hnh 2.22: Chuyn gen bng sng bn gen6.2.2.5.K thut chuyn gen bng sc nhit Dng nhit to ra nhng bin i6.2.2.6. K thut chuyn gen qua ng phn (polen tuber)


31/31

Phng php ny cn c xem l phng php chuyn khng qua nui cy mChuyn gen trc tip thng qua ng phn c nhm Ray Wu i hc Corne(M) bo co thnh cng trn cy la, tip theo cc cng trnh s b vi ADNtng 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 bsinh dc c( tinh t) kt hp vi t bo trng to hp t mang gen ngoi c chuyn vo. Sau , hp t s pht trin thnh ht. Ht ny mm v phtrin 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 non, nhng t bo sinh dc ci cha kp phn chia. Nu lm c nh vy chuyn gen s c thc hin i vi mt t bo sinh sn ci (trng) duy nhtsau khi ti sinh cy s trnh xut hin th khm. Trong th nghim ca Ray Wu, tl ht chuyn gen/ht th nghim l 20%.

chuyen gen vao co the dong va thuc vat

Documents