cndt 2 - animal.pdf

Cng ngh di truyn 2

Chuyn gen ng vt

ng vt bin i gen (VBG) c s dng trongnghin cu chc nng gen in vivo v lm m hnh

nghin cu bnh ngi.

VBG c to ra cc loi ng vt c v, chim, lng c, ng vt khng xng sng.

Chuyn gen ng vt thng c thc hin trnchut nhm to ra cc m hnh bnh ngi.

Gii thiu

Phn tch iu ha biu hin gen t bo ng vt lmt trong nhng hng nghin cu chnh ca sinh hcphn t hin nay.

Kh nng tip nhn DNA ngoi lai ca t bo ng vtc v v s biu hin gen hin din trong on DNAchn vo c pht hin vo nm 1962 bi Szybalskav Szybalski.

- s chuyn DNA thnh cng nh vo s hnhthnh ng kt ta ca DNA vi calciumphosphate; i vo t bo qua c ch thc bo(phagocytosis).

Gii thiu

Ha chuyn nhim (chemical transfection): DNA v

calcium phosphate ng kt ta trn mng t bo v c tip

nhn qua c ch ni nhp bo (endocytosis)

(Primrose and Twyman , 2006)

Cc phng php chuyn gen ng vt

Hnh 1. DNA ngoi lai c a vo t bo

bng phng php ha chuyn nhim

ng kt ta calcium phosphate thng c, mt sloi t bo khng hp thu kt ta rn

cc tc nhn chuyn nhim thay th c phttrin nh cc hp cht polycation (poly L lysine,polyamides, DEAE-dextran)

Mt s hn hp liplid tch in dng hay trung hato phc hp vi DNA. Phc hp ny tng tc vimng t bo v dn n s tip nhn DNA qua c chni nhp bo.

C th c s dng trong chuyn gen mt cch bnvng hay tm thi cng nh chuyn nhng onDNA kch thc ln nh YAC.


Ha chuyn nhim

Hnh 2. Cc hp cht polycation

Phng php chuyn nhim bng xung in cng c s

dng rng ri, mt cch hiu qu trn nhiu loi t bo v in

vivo (da, c hay t bo ung th)

u im: d s dng v kh nng p dng trn nhiu

loi t bo.

Phng php vi tim mang li hiu qu cao nhng i hinhiu cng sc v p dng trn mt s loi t bo nht nh,

nh l giao t ci, trng, t bo phi sm.

Phng php bn gen c th c p dng i vi cc tbo c nui cy hay m cng nh cc ng dng in vivo

(cho t bo da).


Phng php vt l

Hnh 3. Vi nh thhin cu trc mngca t bo hngcu c nglnh cc thiim khc nhau (t),sau khi ctruyn xung in,(a) t= 0,5 ms, (b)2,5 ms, (c) 40ms,(d) 5s, (e) 10s(Chang & Reese,1990).

Hnh 4 Thao tc vi tim thc hin trn hp t giaion sm

DNA khng ti bn, khng c xc nhp s bphn hy, v ch dn n biu hin tm thi

(transient expression) .

mt phn nh ca cc t bo c chuynnhim, DNA c th xc nhp vo b gen t bo

nhn, ti bn v to cc dng t bo bin i gen

bn vng (stable expression). Cc t bo ny c

chn lc bng cc marker.

S xc nhp DNA

Dihydrofolate reductase v kt hp tnh khngmethotrexate.

Cc dng t bo hoang di nhy cm vi Mtx 0.1g/mL,

trong khi cc dng khng (Mtx resistant) c chn lc.

Rodent CAD

CAD protein l enzyme a chc nng tham gia vo cc

bc u tin trong qu trnh sinh tng hp uridine. Mt

trong cc bc ny b c ch bi N-phosphonacetyl- L

aspartate (PALA). Cc t bo ng vt c v sn xut

lng ln CAD t cc bn sao ca gen CAD.

Marker chn lc

XGPRT (xanthine guanine phosphoribosyltransferase):mycophenolic acid resistance

Enzyme t E .coli ny tng t HGPRT ng vt c v.Mi trng chn lc c b sung adenine, mycophenolicacid v xanthine. Hiu qu chn lc tng ln khi b sungthm aminopterin kha con ng ni sinh tng hppurine.

Neomycin phosphotranferase: khng G418

M ha bi Transposon Tn5 v Tn601, th hin tnh khngcc khng sinh aminoglucoside (Kanamycin, neomycin vG418) cc cht c ch sinh tng hp protein, hot ng t bo nhn s v nhn tht.

Marker chn lc

H thng biu hin gen (cassette) ca cc plasmidpSV v pRSV iu khin biu hin gen

chloramphenicol acetyltransferase (CAT) t vi

khun E. coli (Tn9), m ha tnh khng

chloramphenicol, phc v phn tch biu hin gen

tm thi. Protein ny khng hot ng trong t

bo nhn tht.

Luciferase l repoter gen c s dng rng ri cc h thng ng vt v thc vt. Xc tc s oxi

ha luciferin trong phn ng cn kh oxi v ATP

v ta nh sng vng xanh l.

Gen ch th (reporter gene)

chn ging vt nui cn phi chn lc quanhiu th h, tn nhiu chi ph v cng lao ng.

S a vo dng thun mt tnh trng mi c thgy hi/gim nng sut ca con vt v cn nhiu

thi gian ti lp dng thun mi.

S thnh cng ca vic chuyn gen vo t bong vt c v v ca vic cy chuyn nhn sinh

dng vo t bo trng mt nhn cho php thc

hin to dng v tnh ng vt bin i gen.

S to dng v tnh ng vt c v

Gen nhn dng c tim vo hp t

Hp t (c tim) c cy vocon ci hon thnh qu trnh phttrin phi thai

Nhng con vt mang gen nhn dngthnh cc dng di truyn mi

Cc bc to ng vt bin i gen ng vt c v

Phng php chung

Hnh 5. Cc bc tochut chuyn gen.Tin nhn c (malepronucleus) hp t(fertilised egg) c vitim DNA; hp tc bin i cchuyn vo ng dntrng (oviduct) cachut ci mang thaigi (pseudopregnant).Hp t ny s phttrin thnh chutchuyn gen.

Hnh 6. Cc bc tochut chuyn gen bngcch s dng t bo gc.T bo gc phi vi genmc tiu c bin ic vi tim vo phinang (blastocyst). Phinang ny c cy vot cung ca chut mmang thai gi. Phi spht trin thnh chutchuyn gen mang cim di truyn t cc tbo trong phi ban uv t t bo gc.

Thit lp m hnh ng vt phc v nghin cu ccbnh ngi (Phng php gene knock-out)

Sn xut cc protein qu.

V d: s dng tuyn v cho sn xut cc protein dc phm

c trong sa. Sa c th c ti to lin tc, lng ln, qu

trnh tinh ch protein cn thit n gin hn.

Cc ng dng ca ng vt chuyn gen

Vector retrovirus c chuyn vo tbo giai on pht trin phi sm

Vi tim vo nhn nguyn c trngto ca trng th tinh

a cc t bo gc phi ci bin vophi giai on sm, trc khi chuyn vocon ci nhn.

To chut chuyn gen s dng Retrovirus

Retrovirus

http://www.ncbi.nlm.nih.gov/retroviruses/

Hnh 7. Vng i ca Retrovirus

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A483

Hnh 8. Vector retrovirus c v tr

to dng (cloning site) nm trong

vng DNA c ngun gc t

retrovirus. Vector ti t hp c

to ra (mang on chn - mu )

v nhn ln trong t bo E. coli.

DNA ti t hp c a vo t

bo VCV di hnh thc chuyn

nhim. Mt s t bo tip nhn

DNA v to virus ti t hp. Cc

virus ti t hp ny tip tc xm

nhim v biu hin gen cc t

bo mi.

u im

- Rt hiu qu trong vic ci nhp gen chuyn vob gen t bo nhn

- C th p dng trn nhiu loi t bo v trongnghin cu liu php gen.

Hn ch

- Ch chuyn c on DNA kch thc nh(khong 8 kb), do thiu cc trnh t lin quan nqu trnh biu hin gen.

- Khng an ton cho cc sn phm chuyn gen lthc phm

To chut chuyn gen s dng retrovirus

Kch thch con ci rng trng hnglot (35 trng thay v 5 10 trng iu kin bnh thng)

Cho giao phi v thu trng th tinh

Vi tim c tin hnh ngay sau khithu trng. Gen c tim thng dng DNA mch thng.

To chut chuyn gen bng phng php vi tim

ng vt c v, sau khi tinh trng i vo trng,

nhn tinh trng (nhn nguyn c, male pronucleus)

v nhn trng l nhng thc th ring bit. Nhn

nguyn c ln hn nhn nguyn ci, c th phn

bit di knh hin vi.

Hnh 9. Vi nh hunh quang ca trng/phi c th tinh in vitro

(A, B) hay in vivo (C,D) c nhum Hoechst cho thy tin nhn

pronuclei hay nhn (mi tn khng t), cc th cc (u mi tn),

v tinh trng (mi tn t m). Thanh ngang = 50 m. A) Non

vi 2 nhn nguyn v 1 th cc, v tinh trng vng trong sut.

B)Phi giai on 2 t bo vi 2 nhn, 2 th cc v tinh trng. C)

Phi giai on 2 t bo , 2 nhn v 2 th cc. D) Phi giai on

4 t bo vi 4 nhn v 2 th cc.


Hnh 9. Vi nh hunh quang ca trng/phi c th tinh

in- vitro (A, B) hay in vivo (C,D) c nhum Hoechst (http://www.biolreprod.org/content/67/1/256/F1.expansion)

Sau vi tim, 20 - 30 trng c cy vo chut m nuic cha gi (giao phi vi chut c ct ng dntinh). chut, giao phi l phng php duy nht chun b t cung cho vic cy tip theo. 3 tun sau khicy chut m nui s sinh ra chut con t trng ctim vo.

Xc nh DNA chuyn chut con. DNA t chp uichut con c phn tch bng Southern Blot hay PCR pht hin gen chuyn. Chn lc c th mang genchuyn trong t bo sinh dc.


Hn ch

- on DNA chuyn vo gn ngu nhin

- Nhiu bn sao ca gen gn cng mt ch gy biuhin gen qu mc, ri lon sinh l.

Hiu qu

66% hp t sng st sau khi tim

25% hp t pht trin thnh chut con

25% chut con l chut chuyn gen

Trong 1000 trng th tinh c tim, khong 30 35 chut con chuyn gen.


Cc t bo giai on ti phi ca phi chut ang

pht trin c th pht trin trong mi trng nui cy

v c kh nng bit ha (k c pht trin thnh t bo

dng sinh dc) c gi l t bo gc phi

(Embryonic stem, ES)

Tnh nng ca t bo ES khng thay i khi c

dng trong thao tc gen. Gen chuyn c th c ci

vo cc v tr c bit trong b gen ca t bo ES.

To chut chuyn gen bng phng php Ci bin t bo gc phi

Hnh 10 Chuyn gen vo t bo gc phi chuthttp://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A483

1. Thit lp dng t bo gcphi (ES) c nui cy

2. Vector mang gen mc tiuc chuyn vo t bo gcphi c nui cy

3. T bo gc phi mang trnh t gen chuyn trong nhim sc th c timvo phi nang ca chut.

4. Phi nang trn c chuyn vo chutm mang thai h. Chut con th khmc sinh ra, giao phi vi chut thngv sinh ra chut con mang tnh trng qui nh bi gen t t bo gc phi.

Chn lc dng m tnh

H ti t hp Cre loxP xut x t yu t di truynca thc khun th P1.

S ci nhp ca b gen P1 ty thuc vo sn phmca gen cre (circular recombinase/ Cre

recombinase), ct c hiu v ti t hp DNA ca

loxP (locus of crosing over [x] in P1)

K thut ny c s dng nhiu trong nghin cuhu qu sinh hc ca s bt hot gen c hiu m

nhm thit lp m hnh v cc bnh ngi

Bin i gen bng h ti t hp Cre - loxP

Qu trnh to t bo ES vi 1 gen b bt hot.

Thymidine kinase (tk)

Marker chn lc m tnh cho php chn lc cc tbo khng th hin hay mang cc gen marker ny.

M ha bi gen tk virus herpes phosphoryl ha ccdng nucleoside rt hiu qu nh ganciclovir.

Nucleotide c to ra c th c s dng bi DNA

polymerase v ng vai tr nh du hiu kt thc

chui.

Cc t bo biu hin gen tk c th b loi khi nuitrong mi trng cha ganciclovir

Chuyn gen vi cc vector dung lng ln

BAC NST nhn to vi khun

PAC - dn xut t thc khun th P1

YAC NST nhn to nm men

MAC nhim sc th ca ng vt c v

Cc NST ny c th mang DNA b gen t 100 tihn 1000 kb.

Phng php chuyn gen: vi tim.

cndt 2 - animal.pdf

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