VECTO CHUY»‚N GEN

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Chng 1

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

2- 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 vt1. 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

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Hnh 1.1: : To dng bng vector plasmid

4DNA 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.

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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).

6Cc 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).

7V 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.

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A

B

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

9Ngoi 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