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Chng 1

Cc vector s dng trong cng ngh chuyn gen ng vt v thc vt

I. Vector
Trong sinh hc, vector l mt phn t DNA c kh nng mang mt on DNA ngoi lai v khi xm nhp vo loi t bo ch thch hp th c kh nng t ti bn khng ph thuc vo s sao chp ca h gen t bo ch. Ni cch khc, vector l mt phng tin truyn thng tin di truyn trong c th hoc gia cc c th khc nhau. T bo ch thng c s dng l vi khun E.coli. Phn ln cc vector l cc phn t DNA dng vng nh (plasmid) hoc l bacteriophage. Vector c th c ct mt v tr xc nh bng mt enzym hn ch v c ni vi mt on DNA tng hp khc c ct bi cng enzym. Trong to dng phn t, vector l rt cn thit bi v thc t cho thy rng mt on DNA cha gen khng th lm g trong t bo ch. V n khng phi l mt b phn ca genome bnh thng ca t bo, cho nn n s khng c ti bn khi t bo phn chia, khng c biu hin v c kh nng b phn hu kh nhanh. Trong k thut di truyn, vector l cng c c kh nng nghin cu genome ngi v genome cc loi khc v s s dng chng trong nghin cu ang tr nn ngy cng ph bin mt cch rng ri.

II. Cc c tnh ca vector

- Vector phi ln mang DNA ngoi lai nhng khng qu ln. - Vector phi cha cc trnh t kim sot (control sequences) nh khi im ti bn (origin of replication), promoter.

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- Vector phi mang mt hoc nhiu v tr nhn bit ca enzym hn ch. - Vector phi mang cc gen marker chn lc (thng l cc gen khng cht khng sinh). V vy cc t bo cha chng c th c pht hin mt cch d dng.

III. Cc bc trong to dng phn t

- Ni vector v on DNA ngoi lai cn c to dng trong ng nghim to DNA ti t hp nh s xc tc ca enzym ligase. - Bin np DNA ti t hp vo mt dng t bo ch. Chn lc th bin np trn mi trng agar trong a petri c cht khng sinh. - Tch dng DNA ti t hp bng cch s dng mu d (probe).

IV. Cc vector s dng chuyn gen ng vt v thc vt

1. Cc vector s dng chuyn gen ng vt 1.1. Vector s dng thm gen Phn ln cc vector s dng hin nay to ng vt chuyn gen bng cch thm gen c xy dng c hp nht vo genome. Cc phng php ang c s dng hoc nghin cu tng tn s hp nht ca gen ngoi lai hoc duy tr chng nh l cc nhim sc th nh c lp. 1.1.1. Vector thng ti thiu (Minimum linear vectors) i a s trng hp, cc nh nghin cu s dng cc on genome cha mt hoc hai gen hay chun b cc cu trc gen hot ng chc nng t cc yu t khc nhau. Cc on ca vector cha cc vng phin m v iu ha t plasmid. Thc vy, cc vector vng hp nht vi tn s thp hn nhiu so vi cc on DNA thng v trnh t plasmid thng ph hy cc gen chuyn lin kt. iu ny ng i vi cc vector khc nhau nh plasmid, cosmid, phage, BAC v YAC. Tuy nhin mt s nghin cu cho thy rng vector BAC vng hp nht vo genome vi hiu qu ging nh bn sao mch thng ca chng. Ni cch khc, cc vector mang cc on

Hnh 1.1: : To dng bng vector plasmid

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DNA genome di t nhy vi hiu qu cm ca cc trnh t ca prokaryote. iu ny l thch hp nht nh s hin din ca cc yu t cch ly cc on genome di hoc nh mt hiu qu khong cch n gin. Cc on DNA khng cha cc trnh t c bit hp nht vo genome vi tn s tng i thp. Mt s DNA xen vo to ra s ng vt chuyn gen nhiu hn so vi cc DNA khc. iu ny c th xut hin t s c mt ca cc trnh t trong on xen m nhn bit thng xuyn cc trnh t genome (Hnh 1). Mt s cc on xen vo c th cha cc trnh t u tin cho s phin m ca chng v s duy tr ca chng trong phi, tng cng s hp nht xy ra. 1.1.2. Vector cha cc trnh t lp li C ch ca s hp nht c m t hnh 1 bao hm s nhn bit gia cc trnh t ca on xen v ca genome. Tn s ca s hp nht c tng ln nh s c mt c hai u ca cc on xen cc trnh t lp li cao trong genome ch ngay c khi chng b thoi ha nhiu hoc t. b, mt trnh t c mt nhiu tm ng lm tng thm cc on xen tng tn s hp nht. trng hp c bit ny, cc gen chuyn vn khng hot ng. iu ny l do tm ng l vng khng phin m ca genome ph hy gen chuyn. Mt phng php tng t c tin hnh chut, s dng cc trnh t Alu. Cc trnh t ny l cc yu t lp li. Cc trnh t Alu cha 200-300 nucleotid l c nhiu trong genome ng vt c v v c bit l cc vng ln cn hoc trong cc vng phin m. Mt s trnh t Alu c phin m bi RNA polymerase III, lm cho chc nng ca RNA khng r rng v c th khng tn ti. Cc th nghim cho thy rng tn s hp nht c tng ln i vi cc on xen cha trnh t Alu. 1.1.3. Vector transposon Transposonl mt on DNA c kh nng t ti bn mt cch c lp v xen vo mt v tr mi trong cng nhim sc th hoc mt nhim sc th khc (Hnh 1.2). Vi tin b ca k thut di truyn transposon c sa i, thit k thnh cc cng c di truyn vi mc ch c bit.

Hnh 1.2: Cu trc ca transposon

Kch thc ca transposon ni chung l khng di hn 2kb. Nhiu bn sao ca transposon c mt trong genome ti cc v tr ngu nhin mt cch r rng. Transposon c phin m thnh RNA, RNA c phin m ngc thnh DNA si kp. DNA si kp ny hp nht vo genome vi hiu qu cao. S hp nht c iu khin bi gen transposase m ha transposon v cc trnh t lp li o ngc ITR (inverted repeated sequence). Cc trnh t lp li o ngc c mt c hai u ca transposon (Hnh 1.3). C ch ny cho php transposon tri rng ra mt cch nhanh chng v ta khp genome, bao gm c s bt hot gen trong mt s trng hp. S lan ta ca transposon b gii hn bi c ch t bo lm bt hot s phin m ca transposon. Transposon l vector c tim nng i vi s hp nht gen ngoi lai vo genome. lm c iu ny, mt phn ln vng phin m ca transposon b mt i, to ra khong trng i vi gen ngoi lai v ngn cn transposon tri rng mt cch t ch v khng kim sot trong genome. DNA ti t hp cha gen ngoi lai khng c kh nng c bit t hp nht vo genome. S c mt ca gen transposase l cn thit i vi mc ch ny. Tim ng thi transposon mang gen ngoi lai v plasmid vng c kh nng biu hin gen transposase cho php transposon hp nht vi hiu qu c ngha, khong 1-5 % s phi c tim (Hnh 1.3). Protocol ny c p dng trc tin cho Drosophila, s dng transposon P v sau c s dng rng ri to sinh vt chuyn gen (Kayser, 1997). Transposon thy th (mariner) t ra c hiu qu i vi t bo c medaka, g v ng vt c v. Cc sa i khc nhau ca transposon ny lm cho n c th mang mt vector hiu qu v an ton i vi liu php gen (Hackett, 2001).

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Cc vector khc c s dng to cn trng chuyn gen nh Aedes aegypti hoc tm (Tamura, 1999). Gn y transposon c s dng to chut chuyn gen (Dupuy, 2002).

Hnh 1.3: S dng vector transposon chuyn DNA ngoi lai Gen transposase c thay th bng gen mong mun. Transposon ti t hp c tim vo t bo. Vector iu khin s tng hp enzyme gn (intergrase) c cng tim vo. Transposon c hp nht bng cch s dng cc trnh t lp li o ngc ITR ca chng

Trong tt c cc trng hp, transposon v plasmid m ha cho transposase phi c tim vo t bo cht ca phi di cc iu kin khc nhau ty thuc tng loi. V phng din ny, g l khc vi hu ht cc loi khc. Vic tim gen c th c thc hin giai on phi mt t bo m khng th a tr li vo con m nui dng nh trng hp i vi th. Phi tim gen phi c a vo non hong ca mt trng khng mang phi. Sau vi tun p trng di cc iu kin c kim sot tt, g chuyn gen c sinh ra vi mt t l thnh cng c th chp nhn (Shermann, 1998).

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V vy, vector transposon cho php to ra cc ng vt chuyn gen i vi cc loi m vi tim DNA thng thng khng thnh cng. Vector ny cng c xem l an ton. Vector transposon thy th ngay c khi thiu gen transsposae ca n cng c th ti bn v hp nht vo genome ch vi tn s thp. iu ny l do s c mt ca transposase ni sinh ca t bo ch. Vn ny c th gii hn s s dng transposon trong mt s trng hp. Trong khi , transposon BAC ln con c s dng to ra tm chuyn gen n nh hon ton sau mt s th h. Transposon ch c th mang cc on DNA ngoi lai vi chiu di gii hn. Cc cu trc phc tp c s dng biu hin gen ngoi lai r rng cng l mt hn ch. Ngoi ra cc c ch t bo ph hy transposon c th c ch s biu hin ca gen chuyn trong mt s trng hp. 1.1.4. Vector retrovirus a. Cu trc ca retrovirus Retrovirus l loi virus RNA, c v bc bn ngoi. Sau khi xm nhim, genome virus c sao chp ngc thnh DNA si kp, hp nht vo genome t bo ch v biu hin thnh protein. Retrovirus c trng bi chu k ti bn ca chng, c m t ln u tin vo u thp nin 1900 (Ellermann v Bang.O, 1908). Ht retrovirus c kch thc, hnh dng c th thay i i cht nhng ng knh khong 100nm. V ca virus l glycoprotein, to thnh cc gai mng (Hnh 1.4A). Protein trng thnh ny c chia lm hai loi polypeptid (Hnh 1.4B): - Glycoprotein v bn ngoi (SU), khng nguyn ch yu ca virus, c chc nng bm vo th quan. - Glycoprotein mng (TM), bm vo protein SU v, chu trch nhim i vi s dung hp mng.

Hnh 1.4: S cu trc ct ngang ca retrovirus

A. Cu trc ct ngang

B. Cu trc protein v

Bn trong mng l protein c bn (MA), khng nh hnh. Protein ny bao ly capsid (CA). CA l protein phong ph nht trong ht virus (chim khong 33 % trng lng tng s), c hnh khi 20 mt. Bn trong capsid l li, thng c hnh nn, bao gm: RNA genome, protein nucleocapsid (NC), enzym phin m ngc (reverse transcriptase = RT) v enzyme hp nht (intergrase = IN). Genome retrovirus bao gm hai bn sao ca phn t RNA si n, mch thng, c cap u 5 v ui poly A u 3 (tng ng vi mRNA). Genome retrovirus c kch thc khong 811kb. Retrovirus c chia lm hai loi l retrovirus n gin v retrovirus phc tp. RNA ca retrovirus n gin cha 3 nhm gen ch yu: - Gen gas m ha protein li, capsid v nucleoprotein. - Gen pol m ha enzyme phin m ngc v enzyme hp nht. - Gen env m ha protein trong cu trc v ca virus. Trt t ca cc nhm gen ny tt c cc retrovirus l khng thay i: 5 gag pol env 3

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Ngoi ra cn c nhm gen pro m ha enzyme protease viruss. Cc retrovirus n gin bao gm hu ht cc virus gy ung th nh viruss bch cu chut Moloney Mo-MLV (Moloney, 1960), virus ung th tuyn v chut (mouse mammary tumor virus = MMTV) (Bittner, 1936 ). Cc retrovirus phc tp, ngoi ba nhm gen ch yu gag, pol v env cn c cc gen khc nh tat, rev HIV-1. Nhm virus ny bao gm cc lentivirus k c virus HIV-1, spumavirus, HFV (human foamy virus) v SFV (simian foamy virus). mi u ca genome l cc on lp di tn cng (LTR) cha tt c cc tn hiu cn thit cho s biu hin gen ca virus. Mt on LTR gm c 3 vng: U3, R v U5. Vng U3 bao gm cc yu t kim sot s phin m, promoter v gen tng cng (enhancer). y l vng khng m ha c kch thc 75-250 nucleotid, l phn u tin ca genome c phin m ngc, to ra u 3 ca genome provirus. Vng R thng l trnh t c kch thc ngn ch khong 18-250 nucleotid to thnh on lp trc tip c hai u ca genome. Vng U5 l vng khng m ha c kch thc 2001.200 nucleotid to nn u 5 ca provirus sau khi phin m ngc. vng U5 mang v tr poly A v cng vi vng R xc nh s gn thm ui poly A vo. C hai vng U3 v U5 u mang v tr gn att cn thit cho s hp nht genome ca virus vo genome ch. Mt khc, genome virus cn c on dn u (leader), v tr bm primer (primer binding site = PBS) v vng polypurine (polypurine tract = PPT). on dn u khng c dch m, kh di, c kch thc khong 90-500 nucleotid, xui theo v tr khi u phin m, nm gia vng U3 v R, pha u 5 ca tt c cc virus mRNA. PBT di 18 nucleotid b sung vi u 3 ca primer tRNA c hiu c virus s dng bt u phin m ngc. PTT ngn ch khong 10 A hoc G c chc nng khi u s tng hp si (+) trong qu trnh phin m ngc.

Hnh 1.5 : S cu trc genome ca retrovirus

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Ngoi ra cn c cc trnh t cn thit cho s ng gi genome virus gi l trnh t (psi) v cc v tr ct RNA gen env. Mt s retrovirus cha protooncogene. Khi protooncogene b t bin c th gy ra ung th. b. Vector retrovirus Vector retrovirus l cu trc DNA nhn to c ngun gc t retrovirus, c s dng xen DNA ngoi lai vo nhim sc th ca vt ch. Yu t cha kha trong vic s dng retrovirus lm th truyn phn phi gen l s an ton sinh hc (biosafety). Mc ch chnh ca thit k vector l bo m to ra mt virus khng kh nng ti bn (replication incompetent virus) trong t bo ch (Hnh 1.6 ).

Hnh 1.6: Virus nguyn vn c kh nng ti bn v vector retrovirus khng c kh nng ti bn

V nguyn tc, c th to ra vector retrovirus c kh nng ti bn (replication competent retroviral vector) bng cch thm cc trnh t cn thit vo virus (Hnh 1.6). Nhng mt thit k ph bin hn l thay th cc gen ca virus bng cc gen chuyn mong mun to ra vector ti bn khng hon ton (replication defective

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vector). Mt khc, kch thc ca on DNA ngoi lai m vector ti bn khng hon ton c th tip nhn ln hn nhiu so vi vector c kh nng ti bn. S biu hin ca cc protein retrovirus hu ht cc retrovirus gy ung th xy ra t nhin do mt promoter n (single promoter) nm trong on lp di tn cng (LTR) u 5v s biu hin ca cc vng m ha virus phc tp xy ra nh s ct ghp xen k (alternative splicing). Tuy nhin, vic thit k vector l khng b gii hn do s dng promoter retrovirus n. Mt hng thit k khc l s dng promoter phc (multiple promoter) v cc trnh t tip nhn ribosome bn trong (internal ribosome entry site = IRES). S ti np v hp nht gen c hiu qu ph thuc vo cc yu t virus c mt trong vector retrovirus. Mt im lu quan trng khi thit k vector retrovirus l hiu qu ti bn ca virus trong cu trc vector. Phn ln cc vector retrovirus thng c thit k da trn virus Mo-MLV. y l loi virus c kh nng xm nhim vo c cc t bo chut (c th pht trin vector m hnh chut) v t bo ngi (c th iu tr bnh cho ngi). Cc gen ca virus (gag, pol v env) c thay th bng cc gen chuyn mong mun v c biu hin cc plasmid trong dng t bo ng gi. Cc vng cn thit bao gm cc on lp di tn cng u 3 v 5 (3LTR v 5LTR) v trnh t ng gi. Trc y virus h tr kh nng ti bn c s dng ng gi c virus vector v virus h tr. Trong mt phng php tinh vi hn c s dng hin nay, cc dng t bo thao tc di truyn c hiu biu hin cc gem virus do cc promoter khng tng ng c s dng to ra cc virus vector. Cc dng t bo ny c gi l cc dng t bo ng gi hoc cc dng t bo h tr. Chuyn gen v biu hin gen bng mt vector virus c gi l s ti np phn bit vi qu trnh xm nhim ca retrovirus c kh nng ti bn (replication competent retrovirus = RCR). S biu hin ca gen chuyn l do promoter hoc gen tng cng v tr 5 LTR hoc bi cc promoter virus (nh cytomegalovirus, Rous sarcoma virus) hoc bi cc promoter ca t bo (nh beta actin, tyrosine). S nh v chnh xc codon khi u

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ca gen chuyn v cc thay i nh ca 5LTR nh hng n s biu hin ca gen chuyn (Rivire,1995).

Hnh 1.7: Qu trnh ng gi to ht retrovirus

Hnh 1.8: C ch hot ng ca vector retrovirus

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nhn bit cc t bo bin np, cc marker chn lc nh neomycin v beta galactosidase c s dng v s biu hin ca gen chuyn c th c ci tin bng cch thm vo cc trnh t ribosome bn trong (Saleh, 1997). Mt yu cu i vi s hp nht v biu hin ca cc gen virus l cc t bo ch phi phn chia. iu ny gii hn liu php gen tng sinh t bo in vivo v ex vivo, do cc t bo thu nhn t c th c x l kch thch ti bn v sau c ti np vi retrovirus trc khi a tr li bnh nhn. c. ng dng ca vector retrovirus Vector retrovirus l cn thit cho liu php gen, c s dng c hiu qu trong nhiu liu php gen cha bnh cho ngi nh suy gim min dch do thiu ht enzyme ADA, ung th, tiu ng, thiu mu,... Cc vector retrovirus khc nhau c thit k to ng vt chuyn gen v g chuyn gen c bit (Ronfort, Legras v Verdier, 1997). Cc vector ny mang cc trnh t env c kh nng nhn bit cc t bo phi g. Chng c tim vo trng g mi . giai on ny, cc t bo hy cn a nng v c th tham gia vo vic to thnh giao t, dn n truyn gen ngoi lai cho th h sau. Phng php ny cho hiu qu khng cao. Phi giai on ny cha khong 60.000 t bo. Ch mt t l nh cc t bo ny c c hi mang gen chuyn nh vector retrovirus. G chuyn gen to thnh dng th khm v c rt t c hi truyn gen chuyn ca chng cho th h sau. Mt phng php khc chng t c hiu qu hn. Vic tim gen c thc hin giai on phi 16 t bo ti vng ln cn ca cc t bo gc nguyn thy. Cc t bo ny c tim mt cch u tin, to ra cc ng vt chuyn gen c c hi truyn gen chuyn ca chng cho th h sau (Hnh 1.9). Vector retrovirus cng c s dng chuyn gen vo non bo ca b v kh (Chen, 1998, 2001). tin hnh cng vic ny i hi hai iu kin c bit. V ca vector l protein G ca VSV (vesicular somatitis virus) c chc nng nhn bit mng phospholipid v v vy cho php tim vo nhiu loi t bo khc nhau. Cc ht virus c tim vo gia mng trong (zona pellucida) v mng non bo vo lc mng nhn bin mt, to cho genome

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virus c hi tt nht c th n c genome t bo ch (Hnh 1.9). Lentivirus l mt phn lp ca retrovirus. Chng c kh nng hp nht vo genome ca cc t bo pha ngh, c cc t bo tng sinh v t bo khng tng sinh. Kh nng ny c c l do s c mt ca mt tn hiu mt trong s cc protein virus. Protein virus ny nhm ti genome ca virus vng nhn. Lentivirus phc tp hn cc retrovirus n gin, cha thm 6 gen tat, rev, vpr, vpu, nef v vif. HIV l mt loi lentivirus. Vector lentivirus c nghin cu nhiu do tim nng s dng ca chng trong liu php gen.

Hnh 1.9: S dng vector retrovirus to ng vt chuyn gen Genome virus mang gen ngoi lai c a vo t bo tng hp protein virus khuyt. Cc ht virus c s dng tim vo non bo ng vt c v (b, kh), cc t bo mm nguyn thy ca g hoc phi mt t bo ca chut.

Mt nghin cu gn y cho thy rng vector lentivirus tim vo phi mt t bo hoc vi phi c loi b mng trong l c hiu qu c bit i vi vic chuyn gen vo chut (Lois, 2003).

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Cc vector ny ngy cng c ci tin tinh vi hn. Genome ca chng c ngun gc t lentivirus to ra cc ht virus ti t hp c kh nng nhim vo t bo phn chia hoc t bo khng phn chia. Vector ny cha LTR c sa i dn n s t bt hot genome virus hp nht. iu ny lm gim ng k kh nng ti t hp to ra virus xm nhim mang gen ngoi lai vi phng thc khng kim sot. Cc loi vector ny khng c cc gen tng cng. S vng mt cc gen tng cng lm cho n c th thay th cho mt promoter ni sinh iu khin gen mong mun hot ng m khng l thuc vo nh hng ca cc gen tng cng LTR. Vector ny cng cha mt yu t iu ha sau phin m u tin cho vic biu hin gen v mt on ca virus HIV m on ny cho php genome virus i vo nhn ca t bo khng phn chia v hp nht vo DNA ch. Cc vector ny cng c th iu khin s biu hin gen FGFP ca chut, to ra mu xanh hunh quang tt c cc loi t bo hoc ch t bo c khi promoter hot ng tt c cc loi t bo hoc ch t bo c. Lu rng khong 80% chut sinh ra l chut c chuyn gen. Khong 90% s chut ny biu hin gen chuyn ca chng mt s th h. Protein VSV c s dng nh l mt v bc cho php xm nhim hiu qu vo phi. u im chnh ca vector ny l hiu qu bin np cao, thao tc n gin, s xm nhim c th c thc hin bng cch phi loi b mng trong vi th virus. Phng php ny c m rng i vi cc ng vt c v khc m khng c vn c bit. Phng php ny thun li mt cch c bit cho vic chuyn gen vo chut trong khi phng php vi tim l rt kh s dng y. Hn ch ca vector ny cng khng t. Cc ht virus phi c kim sot an ton sinh hc bng bin php thch hp. Bc ny ngy cng c tiu chun ha nhm tng cng s dng cc loi vector ny cho liu php gen. Cc on DNA c kch thc khng vt qu 7-9kb c th c a vo vector ny. S biu hin ca gen reporter l tng i thp trong trng hp ny. Mt khc, s hp nht c lp ca vector xy ra trong cng mt phi dn n ng vt chuyn gen th khm.

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R rng, phng php ny ch c th thay th vi tim trong mt s trng hp. 1.1.5. Vector Adenovius a. Cu trc ca adenovirus Adenovirus l loi virus khng c v, cha genome DNA si kp, mch thng. Trong khi c hn 40 dng adenovirus typ huyt thanh phn ln gy nhim trng ng h hp ngi th ch c nhm ph C typ huyt thanh 2 hoc 5 c s dng lm vector mt cch u th. Chu trnh sng ca adenovirus thng khng lin quan n s hp nht vo genome vt ch, chng ti bn nh cc yu t episome trong nhn ca t bo ch v v vy khng c nguy c pht sinh t bin xen (insertional mutagenesis). Tt c cc ht adenovirus u khng c v, ng knh 6090nm. Chng c tnh i xng icosahedron, d dng nhn thy di knh hin vi in t khi nhum m tnh. V capsid ca adenovirus bao gm 252 capssomer. Li ca ht virus cha ti thiu 4 protein: protein u mt (terminal protein = TP), gn vi u 5 ca genome bng lin kt ng ha tr; protein V (180 bn sao/ht virus) v protein VII (1070 bn sao/ht virus), l cc protein c bn v protein Mu, mt protein nh (4kD), v tr v chc nng cha c bit.

B
Hnh 1.10: Adenovirus A. M hnh cu trc khng gian adenovirus B. Aenovirus nhn di knh hin vi in t

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Hnh 1.11: S cu trc ct ngang ca adenovirus

Genome ca adenovirus l phn t DNA si kp khng phn on, di 30-38kb (cc nhm khc nhau c kch thc khc nhau), c th m ha 30-40gen. C 4 vng gen phin m sm l E 1, E2, E3 v E4, c chc nng iu ha v mt sn phm phin m mun m ha cho cc protein cu trc. Cc trnh t u mt ca mi si l lp li o ngc v vy cc si n bin tnh c th to ra cu trc cn xoong (panhandle). Ngoi ra, cn c protein 55kD lin kt vi u 5 ca mi si bng lin kt ng ha tr. b. Vector adenovirus Vector adenovirus l vector chuyn gen c to ra t cc adenovirus ti t hp. Th h adenovirus ti t hp u tin c thit k bng cch loi b gen E1. S loi b gen E1 lm cho virus khng c kh nng ti bn trong t bo ch. Gen E3 thng cng c loi b dnh ch cho gen chuyn. Phn genome m ha protein cu trc ca virus c thay th bng mt gen marker (nh -galactosidase) hoc gen cDNA liu php. Phn ln cc vector cn xoong c thit k

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gn y ch cha mt trnh t lp li o ngc ITR v mt trnh t ng gi. Tt c cc gen cn thit ca virus c cung cp bi virus h tr. Vector virus ny khng hp nht vo nhim sc th t bo v n tn ti nh mt episome trong nhn. Adenovirus l mt vector chuyn gen an ton, c th xm nhim mt cch hiu qu vo cc t bo khng phn chia v cc t bo ang phn chia ca nhiu loi t bo khc nhau do cc loi t bo ch ny cha cc th quan ph hp vi adenovirus. Khi xm nhim vo t bo ch, phn li mang genome virus v gen ngoi lai i qua l mng nhn. Trong nhn t bo ch, cc gen ca adenovirus v gen ngoi lai s phin m v dch m trong t bo cht to nn cc loi protein cn thit cho virus v protein mong mun do gen ngoi lai m ha. Hn ch ca vector adenovirus th nht l gim kh nng sinh min dch v v tr ngoi nhim sc th ca n lm cho s biu hin gen ch nht thi, khng bn. Kt qu ca mt s nghin cu cho thy rng s biu hin gen chuyn ch ko di t mt vi ngy n mt vi tun.

Hnh 1.12: C ch hot ng ca vector adenovirus

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khc phc nhng vn nu trn, cc th h adenovirus th hai v th ba c to ra. Trong cc vector ny, gen E4 (hoc E2) m ha nhiu protein ca virus c loi b gim s biu hin cc protein virus. c. ng dng ca vector adenovirus Adenovirus ngi th h th nht c s dng rng ri lm vector chuyn gen in vivo (Wilson, 1996). Cc vector ny c s dng chuyn gen ngi in vivo vo t bo mi, t bo phi (CFTR cDNA), t bo mng trong mch, t bo thn (Heikkila, 1996; Sukhatme, 1997), tim, gan, h thn kinh trung ng, c, cc t bo m mu v t bo ung th (Jaffe, 1992; Engelhardt, 1993; Chen, 1994; Chang, 1995; Cussack, 1996; Simon, 1999). Merrrich (1996) so snh tnh ly nhim ca adenovirus ti t hp typ 5 ti bn khng hon ton cc t bo mng trong mch trong mi trng nui cy in vitro. Kt qu cho thy trong mi trng nui cy, s ly nhim l tt c t bo mng trong mch ca ngi v ln. 1.1.6. Vector episome Li th ca qu trnh hp nht khi s dng vector ny l DNA ngoi lai c truyn mt cch n nh cho th h sau. Hn ch chnh l s hp nht him xy ra. Mt vn khc l gen hp nht b ph thuc vo hot ng ca mi trng chromatin ca n, lm thay i thng xuyn s biu hin ca gen chuyn. Mt khc, s lng bn sao hp nht b hn ch, ni chung l khng vt qu 10 v trong mt s trng hp khng phi tt c cc bn sao ny c phin m c hiu qu. Ngi ta ang mong mun loi b phn ln cc hn ch ny khi vector episome. Genome episome c pht hin trong mt s c th sng. y l trng hp i vi vi khun cha nhiu plasmid. Thnh thong cc on nhim sc th c ngun gc t s suy thoi cc b ca nhim sc th c tm thy trong t bo, c bit trong mt s t bo khi u. Cc on nhim sc th ny l cc nhim sc th nh (minute chromosome) c ti bn mt cch c lp v truyn li cho cc t bo con. Cc lp virus khc nhau c genome ti bn mt cch c lp v truyn li cho cc t bo con. y l trng hp i vi cc virus h herpes.

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Genome episome phi cha mt s cc yu t c bn vng. Genome episome c dng vng hoc dng thng. Khi dng vng, thnh phn nh nht ca chng l khi im ti bn. Ti khi im ti bn ny s tng hp DNA c bt u nh l mt h thng lm cho n c th truyn genome ti bn n cc t bo con. Genome episome dng thng phi mang telomere c hai u. Cu trc telomere ny c mt cc u mt ca cc nhim sc th bnh thng. Chng thng xuyn b suy thoi bi exonuclease v c phc hi li nh mt phc hp enzyme c hiu. Telomere bo v nhim sc th khi b suy thoi bi exonuclease. Khi t bo gi hn, s tng hp telomere tr nn t hiu qu hn dn n suy thoi nhim sc th v s cht ca t bo. Cc loi vector episome khc nhau c th c thit k to ng vt chuyn gen. Chng c kch thc tng i nh, ch mang cc yu t ti bn v truyn li cho cc t bo con. Mt phng php khc bao gm s s dng cc on nhim sc th mang tt c cc yu t t nhin chuyn gen cho th h sau. Cc vector episome c bit r v s dng thng xuyn nht l plasmid, cosmid, phage, BAC v YAC. Khng c mt loi vector no c th c s dng eukaryote bc cao do cc yu t ca chng khng hot ng trong cc t bo ng vt. Cc vector ny c s dng ch yu l xy dng DNA v to DNA ti t hp nghin cu v chuyn vo t bo ng vt. Cc vector vi khun ch cha mt khi im ti bn. S bn sao to ra sau khi ti bn DNA l cho php truyn vector c tnh thng k n cc t bo con. Vector YAC dng thng c thit k (Hnh 1.13). YAC cha cc telomere, mt khi im ti bn (ARS 1), mt tm ng (CEN 4), mt gen chn lc (Ura 3) v cc v tr to dng. Vector ny tng i ngn v d thao tc. S d nh vy l do khi im ti bn v tm ng ngn. Khi im ti bn ca nm men Saccharomyces cerrevisae c tp trung trong mt vng genome ngn trong khi Saccharomyces pombe khi im ti bn di n trn vi nghn base. Vector YAC c th mang cc on DNA di 1000kb v chng c th c thit k trong nm men bng ti t hp tng ng. Hn ch ch yu ca vector YAC l khng n nh (Giraldo v Montoliu, 2001).

21

Hnh 1.13: Cu trc ca vector YAC Vector ny cha mt khi im ti bn DNA (ARS 1), mt tm ng (CEN 4) lm cho n c th phn chia vector ti bn v cc t bo con, telomere bo v DNA khi b suy thoi bi exonuclease, hai gen chn lc (TRP 1 v UR 3) v mt v tr to dng m c th a vo mt on c kch thc ln n 1000kb

Vector BAC c thit k ti bn trong E.coli v ch hin din mt bn sao trong mi t bo. Chng c th mang cc on DNA c kch thc ln n 250kb. Chng c th c xy dng trong vi khun bng ti t hp tng ng. iu ny cho php c vic a cc on DNA ngoi lai vo trong vector cng nh loi b chng i. Vector BAC hin l cng c tt nht cho vic chun b cc cu trc mang cc on DNA c kch thc ln dng cho vic to ng vt chuyn gen (Giraldo v Montoliu, 2001) (Hnh 1.14).

Hnh 1.14: S cu trc vector BAC

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Trong t bo ng vt, vn khng th to ra cc vector ngn mang cc yu t t nhin tm thy trong nhim sc th. Khi im ti bn genome ng vt l cc vng xc nh khng tt. Mt s vng c m t c im v c s dng ti bn DNA trong t bo nui cy v trong chut chuyn gen. Nh th on DNA genome cha vi nghn bp l lun c yu cu khi u mt qu trnh ti bn DNA. Mt phng php kh thi l to dng cc on DNA genome vo vector v chn lc cc on c kh nng ti bn cao nht trong t bo ng vt. Th nghim ny cho thy rng ti thiu mt khi im ti bn l c mt trong mt on DNA ng vt c kch thc khong 40kb (Kelleher, 1998). Cc khi im ti bn l khng thch hp cho mt vector vng c truyn mt cch c hiu qu n cc t bo con v th h ng vt con. Mt nghin cu c tin hnh cch y vi nm cho thy rng vector cha mt hn hp cc khi im ti bn ng vt trong mt plasmid c hiu qu cao i vi vic to chut chuyn gen. Vector ny c th c truyn t chut sang vi khun, t vi khun sang phi ln v t phi ln quay tr li vi khun (Attal, 1997). Tuy nhin vector ny khng n nh khi xen mt gen vo trong n. Hn na n khng c duy tr trong sut i sng ca chut v khng c truyn li cho th h con. V vy vector ny cha mt khi im ti bn c hiu qu i vi s truyn li c tnh cht thng k trong qu trnh phn chia t bo nhanh nhng khng c hiu qu trong qu trnh phn chia t bo chm. iu ny c gii thch l do vector ny khng mang bt k yu t no ng vai tr ca tm ng. Nhim sc th eukaryote cha cc trnh t lp li phn trung tm ca chng. Cc trnh t lp li ny bm vo b khung t bo (cytoskeleton) trong sut qu trnh nguyn phn, cho php cc nhim sc th c phn chia v cc t bo con vi phng thc thch hp. Vng tm ng c thm vo mt vi vector episome. Cc on tm ng rt di tt nhin c s dng v chng mang tnh c hiu loi. V vy khng th s dng chng thit k cc vector ngn. Mt s virus c genome dng vng ti bn vi tn s cao trong t bo ng vt v v vy c truyn li cho cc t bo con c tnh cht thng k. y l trng hp ca virus SV40.

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Khi im ti bn ca virus SV40 c nghin cu k. Khi im ny c kch thc ngn nhng cn c khng nguyn T m ho bi genome SV40 v cc thnh phn t bo Linh trng ti bn. Vector SV40 chuyn vo t bo tng hp khng nguyn T (t bo Cos) c s dng ph bin biu hin gen ngoi lai vi tn s cao. Vector ny khng ti bn trong cc t bo khng thuc b Linh trng v v vy khng th s dng to ng vt chuyn gen. Virus herpes trng thi tim sinh trong cc t bo nhim. Mi t bo cha mt hoc vi bn sao genome virus m cc bn sao ny c truyn cho cc t bo con. Trong nhng trng hp nht nh v c bit khi c th b nhim suy gim min dch, genome virus ti bn vi tn s cao gy ra trng thi bnh l c lin quan. y l trng hp i vi virus Herpes simplex, cytomegalovirus, virus Epstein-Barr v mt s virus khc. Cc virus ny c khi im ti bn v mt h thng tm ng gi (pseudocentromeric system). Hai yu t ny c nghin cu mt cch chi tit virus Epstein-Barr (EBV). Cc vng c kch thc nh nht cn thit cho s ti bn ca genome EBV v s di truyn ca chng cho cc t bo con l vng khi im ti bn P (ori P) v gen EBNA 1 (Epstein-Barr nuclear antigen). Vng khi im ti bn P cha hai vng c chc nng ring bit. C hai u bm vo protein EBNA 1. Mt vng ca khi im ti bn P lm ng chc nng khi im ti bn. Vng th hai l cn cho s truyn genome ca virus cho cc t bo con. Cc kt qu nghin cu cho thy rng khi im ti bn ca EBV ch hot ng t bo Linh trng v ch. Khi im ti bn EBV c th b mt v c thay th bng cc on DNA genome ngi hoc cc ng vt c v khc. Mt s cc on DNA genome ny cha mt khi im ti bn cho php vector ti bn v truyn li cho cc t bo con ngay c chut chuyn gen (Kelleher, 1998). Tuy nhin vn cha c bng chng chng minh rng cc vector n nh v truyn li cho th h con. Trong tt c cc trng hp, vector EBV phi mang gen EBNA 1 v vng khi im ti bn P cho protein EBNA 1 ny bm vo. H thng EBV EBNA 1-ori P khng mang tnh c hiu loi. Cc vector cha phc hp EBNA 1-ori P bm khng c hiu

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vo chromatin trong cc t bo eukaryote khc nhau. Mt nghin cu mi y cho thy rng EBNA 1 bm vo ori P v protein EBP 2 nhn ra cc thnh phn cha bit c chromatin (Hnh 1.15 ). Mt vector vng mang vng ori P bm vo EBNA 1 v mt khi im ti bn nm men c duy tr hon ton hiu qu nm men biu hin c cc gen EBNA 1 v EBP ngi (Kapoor, Shire v Frappier, 2001). iu ny cho php a ra gi thuyt l vector episome mang mt khi im ti bn hot ng t bo chut v cc gen m ho protein EBNA 1 v EBP 2 c th c duy tr nh vector vng episome trong sut i sng ca ng vt v truyn li cho th h con. Genome EBV c kch thc 200kb v vector EBV c th mang on DNA ngoi lai c kch thc trn 100kb. Vector cha khi im ti bn SV40 v mt trnh t MAR cng c kh nng duy tr n nh nh cc episome trong t bo nui cy (Jenke, 2002). Cc loi vector ny hin c nghin cu cc phng th nghim l cc vector con thoi (shuttle) v chng mang c h thng ti bn ca prokaryote v eukaryote. Chng c th c truyn n vi khun ng rut (intestine bacteria) v gieo rc vo trong mi trng. iu ny c th trnh c mt cch d dng bng cch loi b khi im ti bn ca prokaryote. Kh nng thit k cc vector c lp khc bao gm s s dng cc on DNA genome di cha cc khi im ti bn t nhin, mt tm ng v cc telomere. Cc vector ny l cc nhim sc th nhn to ca ngi (HAC), ch c th c c sau khi xy ra cc t bin ngu nhin loi b i mt phn ln nhim sc th nhng gi li cc yu t nh nht to ra mt h thng t ch (Vos, 1998; Voet, 2001). Thao tc i vi vector ny khng d dng v rt kh a gen ngoi lai vo trong chng. Hn na, cc on genome di ny cha nhiu gen c th can thip vo sinh l hc ng vt hoc can thip vo gen quan tm khi n c thm vo vector. Nhim sc th chut c kch thc 60Mb bao gm DNA v tinh quanh th trung tm ca chut c to ra trong mt dng t bo lai ng vt gm nhm/ngi. Cu trc ny c duy tr trong mt thi gian di trong t bo nui cy, chut chuyn gen v truyn li cho th h sau (Co, 2000).

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Hnh 1.15: Cu trc ca vector episome vng da trn genome virus Epstein-Barr (EBV) Protein EBNA 1 c m ho bi genome EBV bm vo mt vng khc ca genome EBV. Protein EBNA 1 bm vo mt protein ca t bo l EBP 2. Protein EBP 2 lin kt vi cc yu t khng c hiu ca chromatin. H thng ging nh tm ng ny cho php truyn vector cho cc t bo con. Khi im ti bn ca EBV hot ng hu nh ring bit trong cc t bo Linh trng.

Mt nghin cu cch y vi nm cho thy rng nhim sc th s 2 ca ngi c th c chuyn vo genome chut. N tn ti v truyn li cho th h sau. Nhim sc th s 2 ca ngi c chuyn t nguyn bo si ngi n t bo gc phi chut

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bng dung hp t bo. Cc t bo gc phi mang nhim sc th ngi c s dng to chut chuyn gen th khm. Chut chuyn gen th khm ny biu hin gen globulin min dch (immunoglobulin) nm trn nhim sc th s 2. V vy c th thu c cc khng th n dng ngi t nhng con chut ny (Tomizuka, 1997). Vector episome s hon ton hu dng cho cc nh nghin cu, cho vic nghin cu gen trong cc t bo b nhim cng nh i vi liu php gen v vic to ng vt chuyn gen. 1.2. Vector thay th gen Mt ti t hp tng ng gia hai on DNA c th xy ra trong cc c th khc nhau nu chng mang mt trnh t DNA chung. vi khun kch thc cn thit ca on DNA ny l mt vi trm bp, nm men ti thiu l 20-50bp v t bo ng vt l vi ngn bp gy nn ti t hp tng ng. C s ca c ch ti t hp tng ng c m t hnh 1.16. Nu mt trnh t tng ng n c mt trong genome v DNA ngoi sinh th s ti t hp dn n s hp nht ch (targeted intergration) ca DNA ngoi lai (Hnh 1.17). Nu hai trnh t tng ng khc bit hin din trong DNA ngoi sinh th hai ti t hp tng ng xy ra mt cch c lp dn n s thay th ring bit mt vng genome (Hnh 1.16). V vy mt trnh t ngoi lai c th c hp nht mt cch chnh xc vo v tr cho ca genome. Trnh t ngoi lai ny c th lm ngt qung gen ch, lm cho n tr nn bt hot. Protocol ny c gi l knock-out gen. Trnh t ngoi lai c th l mt gen hot ng c th lin quan hoc khng lin quan vi gen ch. S hp nht ny c gi l knock-in gen. 1.3. Vector sp xp li cc gen ch Ti t hp tng ng trong mt genome l s kin sinh l quan trng xy ra trong cc trng hp khc nhau. S sp xp li genome to ra cc gen globulin min dch hot ng chc nng l mt v d. Tht l quan trng sp xp li genome cc v tr m khng th tin hnh mt cch t nhin vi mt phng thc kim sot.

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Hnh 1.16: S chn lc dng tnh v m tnh kp tch chit t bo m ti t hp tng ng xy ra A. Ti t hp tng ng bao hm s hp nht ca gen neor v loi i gen TK. Cc t bo ny l khng vi G418 v gancyclovir. B. Sau s hp nht ca vector vo mt v tr ngu nhin, cc t bo khng vi G418 nhng nhy cm vi gancyclovir

Hai h thng khng pht hin thy trong gii ng vt c b sung thc hin iu ny. Mt h thng c ngun gc t bacteriophage P1. Genome ny cha cc trnh t LoxP vi kch thc 34 bp, c kh nng ti kt hp v mang tnh c hiu cao ch khi c mt enzyme recombinase ca phage Cre. H thng th hai l tng t mt cch c bn nhng c ngun gc khi u t nm men. Trnh t FRT ti kt hp vi hiu qu cao v mang tnh c hiu khi c mt enzyme recombinase Flp. H thng Cre-LoxP l ph bin nht v c s dng trong nhiu nghin cu khc nhau (Nagy, 2000). Cc th t bin khc nhau ca cc trnh t LoxP, FRT ang c s dng iu chnh hiu qu v tnh c hiu ca s ti t

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hp. Hai loi enzyme recombinase cng tn ti di dng cc th t bin khc nhau. Trong thc t, trnh t LoxP hoc FRT phi c thm vo cu trc ca gen.

Hnh 1.17: S thay th gen bng th t bin s dng ti t hp tng ng kp

u im chnh ca cc h thng LoxP v FRT l s ti t hp ch xy ra khi c mt recombinase. Cc t bo hoc ng vt c vng DNA tip gip vi cc trnh t LoxP th khng c recombinase. Cc enzyme ny c th c b sung vo trong t bo in vitro hoc in vivo ng vt chuyn gen bng cch vi tim trc tip vo t bo cht hoc bng phng php chuyn nhim (transfection method) i vi protein. Gen recombinase c th c

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a vo t bo bng vector adenovirus. Cc vector ny c tim vo m ca ng vt chuyn gen mang vng DNA tip gip vi cc trnh t LoxP. Hn na, plasmid cha cc gen recombinase cng c th c a vo phi giai on mt t bo hoc vo cc t bo soma. Cc plasmid ny c t c hi hp nht do cu trc dng vng ca chng. Chng bin mt nhanh chng trong qu trnh nhn t bo v s c mt ca recombinase l nht thi. Cc recombinase cng c th c truyn cho ng vt chuyn gen bng s chuyn gen. Chut chuyn gen mang gen recombinase c th c lai vi cc dng cha vng DNA tip gip vi trnh t LoxP. Mt cch l tng, cc gen recombinase khng nn biu hin nhiu trng hp trong tt c cc m hoc biu hin thng xuyn. Nu iu ny l cn thit th recombinase c th c t di s kim sot ca cc promoter hot ng trong tt c cc loi t bo. Tri ngc li, cc promoter c hiu i vi tng m c th hn ch s ti t hp LoxP trong t bo m cc promoter ny hot ng. H thng biu hin ny kim sot bi tetrecycline v cc dn xut ca tetracycline, cho php gen recombinase ch c biu hin trong mt loi t bo v ch khi ng vt c tetracycline. Mt bc iu ho khc c th thm vo kim sot recombinase Cre. Cc gen dung hp mang trnh t m ho recombinase Cre v mt phn cc th quan steroid c xy dng. Protein dung hp ny ch hot ng khi c mt steroid v steroid gy ra s bin i hnh th ca protein. Trnh t NLS (nuclear localization signal = tn hiu nh v nhn) cho php recombinase tp trung nhn v t hiu qu ngay c khi nng thp. Trng hp ny c th xy ra ph thuc vo tnh c hiu t bo nhng cc promoter yu ang c s dng biu hin cc gen recombinase. Tt c cc h thng tinh vi ny nhm gy ra s ti t hp c th cng chnh xc ging ht nh s biu hin cc gen t nhin hoc to ra cc iu kin th nghim thch hp nht. iu quan trng l trnh s biu hin cao ca gen ricombinase. Cc enyzme ny bc l mt vi c tnh i vi t bo nng cao, cn hu nh l thch hp nh kh nng nhn bit cc v tr ging nh LoxP hoc FRT trong genome ng vt.

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2. Cc vector s dng chuyn gen thc vt 2.1. Cc vector bin np vo t bo thc vt s dng Agrobacterium 2.1.1. S gy ra cc khi u do Agrobacterium Agrobacterium tumefaciens v A. rhizogenes l hai loi vi khun sng trong t gy ra bnh khi u hnh chp (crown gall) (Hnh 1.18 v hnh 1.19 ) v bnh lng r (hairy root) cc v tr tn thng ca thc vt hai l mm. Ch rt t thc vt mt l mm thuc h Liliaceae v Amaryllidaceae l d b bnh khi u hnh chp. L do gii hn vt ch ny hin nay cn cha c bit. Mt ln khi u, s sinh trng khi u c th tip din khi vng mt vi khun v m khi u c th c sinh trng v trng bng nui cy m trong cc mi trng thiu s b sung auxin v cytokinin ngoi sinh m bnh thng l cn thit xc tin s sinh trng ca m thc vt in vitro. M khi u tng hp amino acid mi v cc dn xut ca ng c bit chung l opine. Loi opine tng hp trong khi u (v d nh nopaline, octopine, agrocinopine, mannopine v agropine) ph thuc vo dng Agrobacterium khi u s hnh thnh khi u. Octopine v nopaline l hai loi opine c ngun gc t arginine v d dng pht hin nht trong m khi u hnh chp. Do nhiu dng Agrobacterium tumefacien ph bin c thit k theo kiu octopine hoc nopaline. Agropine, mt dn xut ca ng c tm thy ph bin trong cc khi u lng r gy ra bi A. rhizogenes. Agrobacterium chu trch nhim i vi s hnh thnh khi u d ha mt cch c chn lc opine m n tng hp c v s dng opine nh l mt ngun cacbon v nit. C vic gy ra khi u v tng hp opine lin quan vi s c mt ca mt loi plasmid c kch thc ln l Ti-plasmid (Tumour inducing =Ti= gy ra khi u) trong t bo vi khun A. tumefaciens v Ri-plasmid (Root inducing = Ri = gy ra lng t) A. rhizogenes.

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Hnh 1.18: Bnh khi u hnh chp cy mm xi (Rubus) do A.tumefaciens gy ra

Hnh 1.19: Cc khi u hnh chp cnh to

Hnh 1.20: S cu trc t bo vi khun Agrobacterium tumefaciens

2.1.2. Ti-plasmid ca Agrobacterium tumefaciens Ti-plasmid (Hnh 1.21A)c tm thy trong tt c cc dng A.tumefaciens gy c, c kch thc khong 200-250 kb. Chng c duy tr n nh trong Agrobacterium nhit di 30oC. Bng phng php lai DNA-DNA v lp bn chui kp d hp (heteroduplex mapping), ngi ta xc nh c Ti-plasmid c 4 vng tng ng. Kt qu phn tch di truyn cho thy vng T-DNA (transferred DNA) v vng gy c (virulence) lin quan n s hnh thnh khi u trong khi hai vng khc lin quan n s tip hp v s ti bn ca plasmid trong Agrobacterium. Trong khi hnh thnh khi u, T-DNA c chuyn vo t bo thc vt v hp nht vi genome nhn. T-DNA n nh trong

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genome nhn. Lai Ti-plasmid vi DNA ca khi u cho thy TDNA trong t bo thc vt l tng ng song song vi T-DNA trong Ti-plasmid ca Agrobacterium. Kt qu ny chng t khng c s sp xp li v tr ca T-DNA trong lc khi u c to thnh. Mt hoc nhiu bn sao ca T-DNA c th c mt cc on lp ni tip. Chng cng c th tch ra v lin kt vi cc vng khc nhau ca DNA thc vt. V tr hp nht ca T-DNA vo DNA thc vt l hon ton ngu nhin. Cc vng tng ng vi T-DNA c tm thy cc Ti-plasmid khc nhau (Hnh 1.21B ). Trong cc dng A.tumefaciens kiu nopaline c s dng ph bin. Vng T-DNA c kch thc khong 24 kb. mt s khi u hnh chp kiu octopine, hai on khng k nhau c tm thy l TL v TR. TL c kch thc 14 kb, c mt trong tt c cc dng t bo bin np v c chc nng tng ng vi T-DNA cc dng t bo nopaline. TR c kch thc 7 kb, c hnh thnh t pha bn phi ca T LDNA trong Ti- plasmid, khng pht hin thy trong tt c cc dng khi u nhng khi s bn sao ca n khc vi TL ngi ta gi thit l xy ra mt qu trnh chuyn c lp. T-DNA c phin m trong cc t bo khi u (Hnh 1.23B), to ra nhiu mRNA polyadenyl. Cc loi sn phm phin m ca TDNA tch ly li l tng i thp so vi cc mRNA ca thc vt khc. Gii trnh t T-DNA kiu nopaline cho thy c 13 khung c m ln (open-reading frame) trong khi TL-DNA v TR-DNA kiu octopine tng ng l 8 v 6. Cc sn phm phin m pha cnh phi ca T-DNA kiu nopaline c chc nng tng ng vi cc sn phm phin m pha cnh phi ca TL-DNA (Hnh 1.21B). Ton b s t chc ca cc gen trn T-DNA v cc vng ln cn l tng t vi cc gen ca genome eukaryote mc d chng khng cha intron. So snh cc trnh t, s pht sinh t bin mt on v gen nhy cng nh s tng sinh ca cc sn phm gen ring l E.coli c s dng pht hin ra chc nng mt s sn phm gen c m ha bi T-DNA. Mt gen trong vng TL octopine m ha enzyme octopine synthase. Ti-plasmid nopaline, cc gen opine synthase bao gm nopaline synthase (nos) v agrocinopine synthase (acs). Trong Ti-plasmid octopine, vng TR m ha hai protein chu trch nhim i vi s tng hp manopine v mt sn phm ca gen xc tc cho s bin i ngc manopine thnh agropine. Locus tmr

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m ha mt enzyme lin quan n h thng cytokinin v s t bin y dn n s tng sinh r t cc khi u hnh chp gy ra mt s loi (cc th t bin r). Cc locus tms 1 v tms 2 lin quan n s tng hp khng iu ha ca auxin v s t bin mt trong hai locus gy nn s tng sinh chi t cc khi u hnh chp nhiu loi thc vt (cc th t bin chi). V vy, T-DNA mang cc gen (tms 1, tms 2 v tmr) m sn phm ca chng khng chu s iu ha bnh thng ca cc con ng chuyn ha thc vt lin quan n s tng hp phytohormone v iu ny gy ra kiu hnh ung th. Do cc gen tms 1, tms 2 v tmr c xem l cc gen ung th. Tuy nhin cn lu rng cc gen c m ha bi T-DNA khng i hi phi chuyn T-DNA vo t bo thc vt v cng khng duy tr s n nh ca n trong genome thc vt. 2.1.3. Ri-plasmid ca Agrobacterium rhizogenes S khi u ca bnh lng t do A.rhizogenes tng t nh s bin np nh A.tumefaciens. Di s kim sot ca vng gy c, hai vng phn tn ca Ri-plasmid c chuyn vo genome thc vt (Huffman, 1984; De Paolis, 1985). Cc vng ny c tn l TL (T-left T-DNA) v TR (T-right T-DNA). TR T-DNA cha cc gen sn xut opine (mannopine hoc agropine) v cc dng c nh r c im nh cc gen opine c trng ca chng (De Paolis, 1985). Mt khc TR T-DNA cn cha hai gen m ha cho s tng hp auxin. Cc gen ny tng ng rt cao vi cc gen auxin ca A.tumefaciens v cho thy c th b sung thm cc dng mang cc gen auxin t bin ca chng (Offringa, 1986). TL T-DNA khng tng ng vi T-DNA ca A.tumefaciens (Huffman, 1984). Ton b TL T-DNA ca c gii trnh t v n cha ti thiu 11 khung c m, tng t vi cc khung c m c tm thy genome ca eukaryote. Chng c promoter v cc yu t polyadenine ha cn thit thc hin chc nng khi chuyn vo trong genome thc vt (Simpson, 1986). Cc gen ny khng tng ng vi bt k gen no

34

Hnh 1.21: Cu trc v s biu hin ca Ti-plasmid kiu octopine v nopaline A. Bn ca Ti-plasmid kiu octopine (pTiAch5) v kiu nopaline (pTiC58) cho thy v tr tng i v kch thc ca cc vng chc nng chnh. Cc v tr t m ch cc vng tng ng ln ca 2 plasmid. //: Cc on lp trc tip 25 bp; CON: vng m ha chc nng tip hp ORI: vng m ha chc nng ti bn v khi im ti bn B. Bn ca vng T kiu nopaline v octopine cho thy cc vng chc nng chnh, cc vng tng ng v cc sn phm phin m. Cc sn phm phin m c bit l: 3(nos): nopaline synthase; 3 (ocs): octonopaline synthase; acs: agrocinopine synthase; 1(tms 1): tryptophan mono-oxygenase; 2(tms 2): indole-3-acetamide hydrolase; 4(tmr): DMA transferase; agr: 3 sn phm phin m cn cho s tng hp agropine.

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bit v cng khng c s tng ng c ngha no i vi TDNA (kiu octopine) ca A.tumefacien. TR T-DNA l khng cn thit i vi s duy tr kiu hnh lng t. Tuy nhin nhiu loi, cc dng Agrobacterium c c TL v TR T-DNA l c nhiu hn so vi cc dng ch mang mt T-DNA (Vilaine v Case-Delbart, 1987). 2.2. C ch chuyn T-DNA Cc vng T-DNA ca c A.tumefaciens v A.rhizogenes u nm bn cnh cc on lp trc tip 25 bp (Hnh 1.11) v cc im kt thc ca T-DNA hp nht trong genome thc vt nm st vi cc trnh t ny. Trnh t lin ng ca bin T-DNA l GGCAGGATATTC/GA/G GT/GTCTAAA/TT/C. Hu ht cc nghin cu c ch chuyn T-DNA t Agrobacterium vo t bo thc vt c tin hnh A.tumefaciens. Vic loi b b phi ca Ti-plasmid kiu nopaline lm mt s hnh thnh khi u, nhng khi on oligonucleotid tng ng vi b phi c to dng vi s nh hng chnh xc vi Ti-plasmid thiu b phi th s hnh thnh khi u li c phc hi (Wang, 1984), cho thy rng cc trnh t lp 25 bp phn cc v tc ng u (cis-acting). S tip xc ca Agrobacterium vi cc hp cht gii phng t m thc vt b tn thng lm cho vng vir ca Ti-plasmid phin m (Hnh 1.22). Trong qu trnh ny mt cht c hot tnh ha hc cao v c trng c nhn bit l acetosyringone. Gen vir A v gen vir C c biu hin vi khun sinh trng sinh dng mc d gen vir C ch c phin m mc thp. Khi Agrobacterium gp dch r ca cc t bo thc vt b tn thng, hoc acetosyringone tinh khit, sn phm ca gen vir A (c th lin kt vi mng) s nhn din v tng tc vi acetosyringone v truyn tn hiu ngoi bo vo trong t bo ny lm hot ha sn phm ca gen vir C. Sau protein vir C bin i hot ha lm cho cc gen vir B, C, D v E khng hot ng v lm tng cng s phin m ca gen vir C. S cm ng gen vir xy ra nh s xut hin cc im t si n trong cc trnh t bin 25 bp nm mp ca T-DNA (Stachel v Zambryski, 1986; Albright, 1987) v s xut hin phn t si n mch thng tng ng vi T-DNA (Hnh 1.22). Cc sn phm ca operon vir D c hot tnh endonuclease (Yanofsky, 1986). Bng mt

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c ch tng t s tip hp ca vi khun, T-DNA c chuyn sang t bo thc vt v xen mt cch n nh vo DNA nhn (Stachel v Zambryski, 1986). S kin ny c th lin quan n protein c m ha bi operon vir E (Winans, 1987). M hnh m t cc s kin xy ra mc phn t trong s tng tc gia t bo thc vt v Agrobacterium hnh thnh khi u hnh chp c trnh by hnh 1.22. 2.3. Plasmid Agrobacterium l vector bin np Kh nng chuyn mt cch t nhin cc trnh t DNA xc nh vo genome thc vt ca Agrobacterium c s dng vo vic pht trin cc vector bin np thc vt. Cc vector ny li dng mt s c tnh bm sinh ca Agrobacterium l qu trnh bin np trung gian (mediated transformation process). Vo u thp nin 1980, mt s nhm nghin cu Ti-plasmid loi b tt c cc gen onc ca T-DNA. Khm ph quan trng th nht l cc gen onc ny khng cn thit i vi vic chuyn T-DNA vo t bo thc vt v khng hp nht vo DNA nhn. V vy cc gen ny c th c thay th. N khng ch cho php xen DNA ngoi lai vo m cn cho php loi b cc chc nng ca gen onc. Tuy nhin cn lu rng, nos hoc ocs l cc gen marker hu ch i vi s bin np bi v hot tnh enzyme ca cc sn phm gen ca chng c th c pht hin bng mt xt nghim n gin. Cho n nay, gii hn kch thc ca on DNA xen vo cha c cng b. S kin quan trng th ba l s khm ph cc sn phm ca gen vir cn hot ng chc nng trong trans. Cui cng, T-DNA khng gy ung th c mt trong ton b thc vt ti sinh c truyn li cho th h sau theo kiu di truyn Mendel. 2.4. Cc thnh phn c bn ca vector Ti-plasmid khng gy ung th c im ca chuyn gen qua trung gian Agrobacterium l bt k DNA no to dng u c th c chuyn vo genome ca t bo thc vt hai l mm. DNA ngoi lai phi c nm mp cc trnh t bin ca T-DNA v duy tr n nh trong dng Agrobacterium mang nhm b sung y ca cc gen vir dng cis hoc trans (nh v trn plasmid h tr gy c phn tn). Cc gen trn nhim sc th ca Agrobacterium kt hp vi vng gy c

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c m t v c lin quan vi vic vi khun nhn ra mt thnh phn ca b mt t bo thc vt, ri gn vo sau .

Hnh 1.12: S tng tc gia Agrobacterium vi t bo thc vt v c ch chuyn T-DNA

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nhn ra cc t bo c bin np, cc gen tri khng thuc khng sinh ca vi khun c xen vo trong cc trnh t lp 25 bp di s kim sot ca T-DNA promoter v cc tn hiu polyadenyl ha. Cc gen khng thuc khng sinh dng khm nh th biu hin mt cch c hiu qu trong t bo thc vt bt k mi trng di truyn no. Vic lin kt mt cch cht ch cc gen marker vi DNA ngoi lai c 2 li ch: th nht l chn lc trc tip cc m thc vt c bin np DNA ngoi lai vo mt cch chc chn; th hai l m bo DNA ngoi lai trong cc dng bin np c trng chn lc khng xen vo vng genome khng c phin m. 2.5. Cc vector bin np thc vt khng gy ung th da trn Tiplasmid C A.tumefaciens v A.rhizogenes u c s dng chuyn DNA ngoi lai vo t bo thc vt. Nhiu vector bin np da trn Ti-plasmid c pht trin (Bng 1.1 v bng 1.2). Cc vector ny khng cha bt k trnh t ung th no v v vy t bo thc vt c th sinh trng bnh thng sau khi chuyn DNA vo nhn ca n. Cc vector khng gy ung th hin ang s dng c th c chia lm hai loi l cis v trans, da vo vic c hay khng cc vng T-DNA nm mp cc trnh t lp trc tip 25 bp trn cng n v ti bn (replicon) nh cc gen vir hoc trn mt plasmid phn tn (Hnh 1.13). Trc y, loi vector thng c cp n l vector lin hp (co-intergrative vector), tuy nhin gn y vector nh th (binary vector) l ph bin hn. 2.5.1. Cis vector y l cc vector c ngun gc t Ti-plasmid kiu di m gen onc trn T-DNA c loi b v trong mt s trng hp c thay th bng mt on DNA c hiu c vng tng ng vi mt vector to dng nh m ch c th ti bn E.coli. Chin lc vector ny ph thuc vo s lin hp trong A.tumefaciens gia cc vng tng ng trn Ti-plasmid sa i (h tr mang gen vir) v mt vector to dng nh E.coli (vector trung gian) mang gen marker

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40

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chn lc s hot ng chc nng trong cc t bo thc vt v cc trnh t duy nht cho vic xen DNA ngoi lai vo. Vector trung gian mang trnh t DNA ngoi lai thng c a vo A.tumefaciens bng s tip hp v s dng s chn lc thch hp s thu c th nhn tip hp vi DNA ngoi lai n nh trong T-DNA l kt qu ca ti t hp tng ng (Hnh 1.23A ).

Hnh 1.23: S h thng vector lin hp (A) v vector nh th (B) VIR: vng gy c; HOM: cc vng tng ng trong s ti bn c th xy ra i vi s lin hp; LB: bin tri; RB: bin phi; MCS (multicloning site): cc trnh t to dng; PTM: marker bin np thc vt; RES: marker khng thuc khng sinh chn lc i vi s c mt ca cc trnh t vector trong vi khun ch; oriT: khi im chuyn; ColE1: khi im ti bn t plasmid ColE1; RK2: vng c ph khi im ti bn rng, c ngun gc t pKR2

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mt s vector nh pGV3850 (Hnh 1.24), c hai bin ca T-DNA l trn Ti-plasmid sa i. Trong cc vector nh pGV2260 (Debleare, 1982), cc trnh t lp 25 bp l trn vector trung gian, trong khi vector pTiBS3-SE (Fraley, 1985), bin phi l trn vector trung gian v bin tri trn gen vir ca plasmid. Bt k u trong cc v tr khi u ca cc trnh t lp 25 bp, kt qu thc sau khi lin hp l s xen cc trnh t DNA ngoi lai bin T-DNA lp li trn Ti-plasmid sa i. V vy cc dng A.tumefaciens mang cu trc ny, T-DNA c chuyn vo genome thc vt trong sut s bin np l kt qu hot ng ca vng vir dng cis.

Hnh 1.24 : Cu trc v s s dng vector lin hp pGV3850 Bn cho thy cu trc ca vector bin np thc vt pGV3850 v vector trung gian pGV1103 v kt qu ca vic hp nht pGV1103 vo pGV3850. LB: bin tri; RB: bin phi; Pnos: promoter nopaline synthetase; npt-II: trnh t m ha neomycin phosphotransferase-II t Tn5; ocA: trnh t polyadenyl ha octopine synthase; E: EcoRI; H: HindIII; B: BamHI; nos: nopaline synthase; ApR: gen khng ampicillin; KmR: gen khng kanamycin

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Mt v d chi tit v vector cis l pGV3850 (Zambryski, 1983). Vector ny c loi b cc gen onc ca Ti-plasmid kiu nopaline (C58) v thay bng pBR322 (Hnh 1.25). Bt k trnh t DNA ngoi lai no c to dng trong pBR322 u c th a vo pGV3850 bng qu trnh chuyn vo Agrobacterium theo cch tip hp ti t hp bao gm hai bc. Trc ht pBR322 mang gen cn chuyn v mt marker khng thuc khng sinh cho php chn lc Agrobacterium (kanamycin hoc streptomicin/spectinomycin) c a vo dng Ecoli cha hai plasmid h tr pGJ28 v pRGdrd11. Cc plasmid ny cung cp ColE1 c chc nng h tr, cho php chuyn c 3 plasmid vo Agrobacterium qua tip hp. Tuy nhin pBR322 khng th ti bn trong Agrobacterium v v vy n s khng c bo tn, nhng s ti t hp c th xy ra gia pBR322 v pGV3850 lm cho gen quan tm c chuyn vo Tiplasmid. Th nhn tip hp c th c chn lc nh tnh khng do plasmid m ha v tnh khng rifampicin do nhim sc th ca Agrobacterium m ha. 2.5.2. Vector nh th Vector trans hoc vector nh th c da trn cc plasmid c th ti bn c E.coli v Agrobacterium v cc plasmid ny c cha cc trnh t bin ca T-DNA (Hnh 1.23B). Cc vector ny c th c thit k sao cho cc trnh t bin cnh MCS cho php xen DNA ngoi lai vo v cc marker cho php chn lc trc tip cc t bo thc vt c bin np. Plasmid c th c thao tc trong E.coli v c chuyn vo qua s tip hp vi cc dng Agrobacterium mang plasmid c cha vng vir nhng thiu T-DNA v cc trnh t lp 25 bp. Cc plasmid nh th thng l cc th t bin mt on n gin ca Ti-plasmid octopine kiu di hoc kiu nopaline (Bng 1.2). Vic chuyn DNA ngoi lai trn vector to dng vo t bo thc vt c th c thc hin do hot ng chc nng ca vng vir. pBin19 l mt vector nh th ph bin da trn n v ti bn (replicon) vt ch m rng ca pRK252 (Hnh 1.25A). V vy n c th ti bn trong c E.coli v Agrobacterium, cho php xen DNA ngoi lai vo vector v sng lc trong E.coli trc khi chuyn vo Agrobacterium. Vector ny cha marker khng kanamicin (Kmr)

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Hnh 1.25 : Cu trc v s s dng vector nh th pBin19 A. Bn ca vector nh th pBin19 LB: bin tri; RB: bin phi; lac: vng b sung ca operon lac; Pnos: promoter gen nopaline synthetase; npt-II: trnh t m ha neomycin phosphotransferase-II t Tn5; nosA: trnh t polyadenyl ha gen nopaline synthase. B. S xm nhp ca pBin19 vo A.tumefaciens LBA4404 s dng plasmid h tr pRK2013 v sau loi b plasmid h tr trong Agrobacterium.

gip chn lc trc tip trong vi khun cng nh cc trnh t bin T-DNA cnh mt marker chn lc tri vi mc ch s dng trong

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cc t bo thc vt (mt gen khng kanamicin nm cnh promoter nos v trnh t poly(A) thm vo) v mt MCS trong vng b sung ca -galactosidase. S sng lc cc th ti t hp c th c tin hnh Lac- E.coli nh s b tr vi s c mt ca IPTG v XGAL (Groneborn v Messing, 1978). Vector ny c th c a vo Agrobacterium bng s tip hp s dng cc chc nng h tr ca pKR2013. Agrobacterium ch LBA4404 cha Ti-plasmid (pLBA4404) loi b T-DNA nhng vng vir vn khng i. Th nhn tip hp c th c chn lc nh tnh khng kanamicin v rifampicin. V d v h thng vector nh th c trnh by bng 1.2. Cc vector nh th ny khc nhau v kch thc, s ti bn n nh trong Agrobacterium, cc v tr to dng, s b tr c th sng lc plasmid ti t hp trn cc a X-GAL, cc gen marker chn lc cc thc vt bin np v cc gen marker chn lc cc th nhn tip hp, nhng phn ln c bit l thch hp vi mt s plasmid h tr mang gen vir ca A.tumefacien cng nh vi nhiu Riplasmid. 2.6. Vector bin np thc vt s dng A.rhizogenes Cc vector ny c s dng ln u tin khi bin np vo cc loi thc vt m s ti sinh ton b c th t cc t bo n l l kh khn. mt s loi, s ti sinh c th c th xy ra t s nui cy r gy ra bi A.rhizogenes thng qua s pht trin phi soma hoc s pht sinh c quan. C ch kiu hnh lng r b c ch lm pht sinh hnh thi chi hin nay cha r. Thc vt ti sinh t lng r thng b bin i hnh thi: l b gp np, h thng r n nghing, sinh trng cn ci v kh nng sinh sn km. 2.6.1.Vector lin hp y l vector trung gian c thit k cho php thao tc E.coli v cha vng T-DNA ca Ri-plasmid. S dn nhp vector vo A.rhizogenes mang mt Ri-plasmid bnh thng lm n nh cc trnh t ca vector trung gian l kt qu ca ti t hp tng ng ni phn t trong Ri-plasmid (Jensen, 1986).

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2.6.2.Vector nh th Vector nh th loi b ht kh nng gy hi c ngun gc t A.tumefaciens (Bevan, 1984; Van den Elzen, 1985) c th a vo A.rhizogenes v s ti bn n nh min l s chn lc c duy tr. Sau nhim A.rhizogenes mang vector nh th vi thc vt s lm chuyn T-DNA t vector nh th cng vi T-DNA ca A.rhizogenes vo genome thc vt (Shalin, 1986; Simpson, 1986). Cc gen ny khi chuyn vo genome thc vt c nh v gia cc trnh t bin ca vector nh th. Cc trnh t bin ca vector nh th ny c tc dng sau khi s cm ng ca vng vir ca Ri-plasmid kiu di ngay ti ch (Simpson, 1986; Trulson, 1986). iu ny c th xy ra l do tng ng ln, mc DNA, gia cc vng gy c (virulence region) ca A.tumefacien v A.rhizogenes (Huffman, 1984). Gn y lng t (hairy root) c chua v da chut c to ra bi A.rizhogenes kiu di mang vector nh th vi marker chn lc Kmr. Th ti sinh t mi trng nui cy lng r khng kanamicin ny c sng lc i vi thc vt kiu hnh bnh thng v mt s c phc hi m thiu T-DNA ca A.rizhogenes nhng c mt T-DNA ca vector nh th (Shalin, 1986; Trulson, 1986) l do s chuyn c lp ca cc T-DNA tch ri. c tnh ny ca A.rhizogenes lm cho n tr nn hp dn nh l mt vector bin np nu nh qu trnh ny c th c m rng i vi nhiu loi cy trng khc.

Ti liu tham kho chnh

Makrides SC. 2003. Gene Transfer and Expression in Mammalian Cells. Elsevier Science B. V. USA. Louis-Marie H. 2003. Animal Transgenesis and Cloning. John Wiley and Sons, Ltd. USA. Leland HH, Leroy H, Michael LG, Ann ER, Lee MS, Ruth CV. 2000. Genetics, The McGraw-Hill Companies, Inc. USA. Winter PC, Hickey GI, Fletcher HL. 1998. Instant Notes Genentics. Printed by Biddles Ltd, Guildford. UK.

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Glick BR, Jackj P. 1994. Molecular Biotechnology. ASM Press. Washington D.C. USA. Chopra VL, Anwan N. 1990. Genetic Engineering and Biotechnology. Oxford and IBH Publishing CO.PVT, Ltd. UK. John D, Roderick S, Philip A, Richard W. 1988. Plant Genetics Transformation and Gene Expression (A Laboratory Manual). Blackwell Scientific Publications. UK.