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Transcript of Chuy»ƒn gen eGFP » g 

  • 1. Sinh vin thc hin 1. Mai Hu Phng 2. Nguyn Duy Hi 3. Ng Th Hoi Dim 4. L Th Thu Trang 5. Nguyn Thanh Nh 6. L Th Hng Friday, November 14, 2014 1

2. Quy trnh v cc phng php chuyn gen ng vt M hnh chuyn gen g Th nghim chuyn gen GFP trn G (Gallus Gallus domesticus) 1 2 3 Friday, November 14, 2014 2 3. Friday, November 14, 2014 3 1.1. Quy trnh chuyn gen ng vt 4. 1 2 3 5 6 Chuyn gen vo tinh trng Chuyn gen qua liposome Phng php vi tim Chuyn gen nh virus Chuyn gen nh xung in 4 Chuyn gen nh s dng t bo gc phi Friday, November 14, 2014 4 1.2. Phng php chuyn gen ng vt 5. 2.1. Gii thiu Qu trnh chuyn gen ng vt c tin hnh thnh cng trn nhiu i tng khc nhau. c bit, g, do cu to v sinh l c nhiu u th tin hnh chuyn gen. y chnh l i tng tuyt vi sn xut protein ti t hp trong trng. y l mt bc quan trng tin n mc tiu nhn ging g mi c th nhng qu trng vng mang protein dc phm. Friday, November 14, 2014 5 6. 2.2. Nhng thun li v kh khn khi s dng g lm i tng chuyn gen Thun li Thi gian th h ngn, tc sinh trng nhanh. Nng sut trng cao. Kch thc thch hp Trng c mi trng v trng t nhin. TP protein n gin. S di c ca t bo mm. Friday, November 14, 2014 6 7. 2.2. Nhng thun li v kh khn khi s dng g lm i tng chuyn gen Kh khn Trng g ngay khi va c ra, phi bt u pht trin v cha khong 40.000-60.000 t bo. Trng ln, d v v kh thao tc. Kh tip cn giai on phi sm ca chng. Friday, November 14, 2014 7 8. 3.1. i tng nghin cu Sn lng trng mt nm khong 250 - 270 qu. Trng to, nng 60 - 65 g. T l lng trng so vi lng cao. V trng mu trng, d kim tra phi trong qu trng p. G Leghorn l ging chuyn trng cao sn nht th gii hin nay: Friday, November 14, 2014 8 9. 3.1. i tng nghin cu G Leghorn l ging chuyn trng cao sn nht th gii hin nay G Leghorn l i tng rt thun li cho cc nghin cu chuyn gen gia cm vi mc ch sn xut protein dc liu trong lng trng trng. Friday, November 14, 2014 9 10. 3.2. Vector pT2/BH-CVpf-SB11 Friday, November 14, 2014 10 Vector transposon Sleeping Beauty (SB) H thng transposon SB cha mt gen n m ha cho enzyme transposase v mt transposon cha u tn cng lp li ti v tr lin kt vi transposase. 11. 3.2. Vector pT2/BH-CVpf-SB11 Friday, November 14, 2014 11 12. Friday, November 14, 2014 12 Nhn dng vector pT2/BH- CVpf-SB11 To phc hp lipoplex (ADN- liposome) Chuyn gen bng PP vi tim Chuyn gen qua tinh trng Kim tra s c mt ca gen cn chuyn 13. 3.2. Nhn dng E.coli mang vector pT2/BH-CVpf-SB11 Vector pT2/BH-CVpf-SB11 c bin np vo E.coli nh xung in Nui cy vi khun E.coli mang gen bin np. Tch chit, thu nhn plasmid t E.coli. Plasmid s c in di kim tra trn gel agarose 0.8 %. Friday, November 14, 2014 13 14. Friday, November 14, 2014 14 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 15. 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 3.4.1. Phng php rn kim tim ng mao qun thy tinh, d = 1 mm My ko kim ko c u kim tim c ng knh 30 40 8 m B u nhn c chiu di v vt ph hp Friday, November 14, 2014 15 16. 3.4.2. Phng php to phc hp lipoplex (ADN- liposome) Lc nh LipofectaminTM 2000, nh 2 l ln b mt ca 100 l mi trng Opti-MEM. 5 pht nhit phng. Nh 0,8 g ADN ln v tr nh LipofectaminTM 2000 trn. 20 pht nhit phng to thnh phc h ADN- Liposome. Friday, November 14, 2014 16 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 17. 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 3.4.3. Phng php m ca s Chun b v y ca s V vi Dng my khoan mini khoan v trng thnh cc mnh trn c ng knh 1 cm. Ra sch bi vi cn 70o ra bng PBS. Bo qun trong PBS s dng cho cc TN. Friday, November 14, 2014 17 18. Chun b v y ca s Mng v Tch mng v ra khi mng vi. Ra nh mnh v vi v bo qun trong PBS dng cho cc TN sau. Friday, November 14, 2014 18 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 3.4.3. Phng php m ca s 19. M ca s kt hp vi tim DNA Friday, November 14, 2014 19 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 3.4.3. Phng php m ca s 20. M ca s kt hp vi tim DNA Trng g c kh trng bng cch xng KMnO4 + formon v t nm ngang trc khi tin hnh vi tim t nht 2h. Dng my khoan mini ct mt vng trn nh c ng knh 0,5 cm trn phn v vi ca trng. Friday, November 14, 2014 20 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 3.4.3. Phng php m ca s 21. M ca s kt hp vi tim DNA Bi paraffin xung quanh mp ca s. Gp phn v vi khoan ra v nh 1 git PBS ln ca s. Trng c a vo bung thao tc v trng v c x rch mng v bng panh. Friday, November 14, 2014 21 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 3.4.3. Phng php m ca s 22. M ca s kt hp vi tim DNA S dng kim vi tim tim 2 l ADN vo xoang di phi (ch khng bt kh i vo phi). Friday, November 14, 2014 22 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 3.4.3. Phng php m ca s 23. M ca s kt hp vi tim DNA Ly 1 ming mng v y ln ca s. y tip 1 mng vi ln mng v y. Hn kn ca s bng keo Duco. Trng c em i p v t theo chiu thng ng vi u t quay ln trn. Friday, November 14, 2014 23 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 3.4.3. Phng php m ca s 24. 3.4. Chuyn gen GFP bng vi tim vo phi G 0 gi p 3.4.4. Phng php p trng Trng 0 gi p s c xng kh trng bng KMnO4 v focmon. Trng sau khi kh trng c xp vo khay vi u t quay ln trn. Khay trng c chuyn vo t p vi ch p nh sau: Nhit : 37,8 oC. o trng: 2h/ln. 10-12 ln/ngy. Thng thong: vn tc gi 77 cm/giy; tc qut: 300 vng/pht. Friday, November 14, 2014 24 25. Friday, November 14, 2014 25 3.5. Chuyn gen GFP bng PP chuyn gen qua tinh trng 26. Friday, November 14, 2014 26 3.5. Chuyn gen GFP bng PP chuyn gen qua tinh trng 3.5.1. Phng php ly tinh trng g Thc hin thao tc gy xut tinh g trng cong ui ln ngn ci v ngn tr bp nh vo vng l huyt v hi n vo bng di l huyt tng thm kch thch. Dng mt tay vn ngc ui g ln l vng huyt hng tinh dch. 27. Friday, November 14, 2014 27 3.5. Chuyn gen GFP bng PP chuyn gen qua tinh trng 3.5.2. Phng php xc nh nng tinh trng Pha long tinh dch: Dng ng trn hng cu ht tinh dch nguyn n vch 0.5, sau ht tip dung dch NaCl 3% ( git cht tinh trng) n vch 101, lc nh trong 2 - 3 pht trn u dung dch. B 3 - 4 git u, ri nh 1 git ln bung m hng cu, y lamen. 28. Friday, November 14, 2014 28 3.5. Chuyn gen GFP bng PP chuyn gen qua tinh trng 3.5.2. Phng php xc nh nng tinh trng Kt qu s lng t bo c tnh theo cng thc: 7 10. 80 4000000.200. n n C Trong : C: Nng tinh trng (tinh trng/ml) n: S lng tinh trng c trong 80 vung nh 200: S ln pha long tinh dch 4.000 000: Th tch 1 vung nh:1/4000000 cm3.(ml) 80: Tng s vung nh m. 29. 3.5.3. Phng php to phc hp lipoplex (ADN- liposome) Lc nh LipofectaminTM 2000, nh 20 l ln b mt ca 100 l mi trng Opti-MEM. 5 pht nhit phng. Nh 8 g ADN ln v tr nh LipofectaminTM 2000 trn. 20 pht nhit phng to thnh phc h ADN- Liposome. Friday, November 14, 2014 29 3.5. Chuyn gen GFP bng vi tim vo phi G 0 gi p 30. Friday, November 14, 2014 30 3.5. Chuyn gen GFP bng PP chuyn gen qua tinh trng Ly 1-1,5 ml tinh dch thu c pha long vi 4 ml mi trng DMEM khng c FBS (t l pha long thch hp nht ca tinh dch v mi trng l 1:3). Trn u phc hp lipoplex vi tinh dch va thu c (tng khong 6ml), 20 - 50 pht ri tin hnh TTNT cho g mi. 3.5.4. Phng php chuyn phc hp lipoplex vo tinh trng 31. Friday, November 14, 2014 31 3.5. Chuyn gen GFP bng PP chuyn gen qua tinh trng 3.5.5. Phng php th tinh nhn to 32. 3.6. Phng php thu nhn DNA t g mang gen chuyn Phng php tch t bo phi 7 ngy p v nui cy nguyn bo si Friday, November 14, 2014 32 Phng php thu nhn DNA t t bo hng cu v ni quan g 33. 3.6.1. Phng php tch t bo phi 7 ngy p v nui cy nguyn bo si Tch t bo phi 7 ngy p Kh trng trng bng cch lau cn 70 . Ly phi ra khi trng v cho vo a petri ng dung dch mui sinh l. Ra sch 2, 3 ln bng dung dch sinh l cho sch mu, lng trng v lng trng. Friday, November 14, 2014 33 34. Tch t bo phi 7 ngy p Chuyn phi vo bung thao tc v trng v ra bng PBS khong 2 ln. Chuyn sang ng Eppendorf, ct nh trong 0,5ml trypsin 0.25% trong 2 pht. Thm 1 ml mi trng nui cy bt hot Trypsin. Loi b cn to, thu dch t bo v dng nui cy. Friday, November 14, 2014 34 3.6.1. Phng php tch t bo phi 7 ngy p v nui cy nguyn bo si 35. Nui cy n dng m s lng t bo trong dch huyn ph. Ht dch huyn ph t bo chia vo 4 ging c sn 0,5 ml MT nui cy. Sau 24 gi, kim tra v thay mi trng cho cc ging nui cy. Sau 72 gi, cy chuyn v nui cy in vitro mt t bo > 90% v tin hnh sng lc puromycin kim tra s c mt ca gen chuyn. Friday, November 14, 2014 35 3.6.1. Phng php tch t bo phi 7 ngy p v nui cy nguyn bo si 36. Sng lc t bo mang gen chuyn bng khng sinh puromycin Stock Puromycin (10 g/ml) 1 g/ml, 2 g/ml v 3 g/ml. Friday, November 14, 2014 36 0 g/ml 3 g/ml C TN1 TN2 TN3 TN4 TN5 3.6.1. Phng php tch t bo phi 7 ngy p v nui cy nguyn bo si 37. Sng lc t bo mang gen chuyn bng khng sinh puromycin Sau 24 gi - 48 gi nui cy, mt t bo t khong 0,5x106 t bo/ging, x l bng puromycin trn 2 l: l TN v l C. Sau khi b sung puromycin vo cc ging nui cy, lc nh puromycin ho ln vi mi trng v tip tc nui trong t m 37oC, 5% CO2. Sau 24h b sung puromycin, mang mu quan st v m s lng t bo. Friday, November 14, 2014 37 3.6.1. Phng php tch t bo phi 7 ngy p v nui cy nguyn bo si 38. m s lng t bo Pha long huyn ph t bo 200 ln bng PBS sau dng pipet nh 1 git ln bung m hng cu, y lamen. Nng t bo c tnh theo cng thc sau: Friday, November 14, 2014 38 C = 80 000.000.4200xxn = n x 10 x 106 C - nng t bo (t bo/ml dch mu t bo) n - s lng t bo m c (trong 80 nh) 200 - s ln pha long mu dch t bo 4.000.000 - th tch ca 1 con tnh bng cm3 (ml) 80 - tng s con c m 3.6.1. Phng php tch t bo phi 7 ngy p v nui cy nguyn bo si 39. Ph v t bo T bo sau khi ly t cc ging nui cy s c li tm thu cn t bo. T bo trong cc ging c cho vo khong 10 dung dch TE. hn hp t bo trong TE vo nc 94oC trong khong 10 pht. Friday, November 14, 2014 39 3.6.1. Phng php tch t bo phi 7 ngy p v nui cy nguyn bo si 40. Thu nhn DNA t t bo hng cu Ly mu g 10 ngy tui. Ly tm vi tc 1200 RPM, trong 15 pht. Loi b huyt tng, thu hng cu. B sung thm 0,5 ml TE. Sau s dng b kit tch ADN. Friday, November 14, 2014 40 3.6.2. Phng php thu nhn DNA t t bo hng cu v ni quan g 41. Thu nhn DNA t ni quan g G con khong 1 thng tui. Tch ni tng ca g (gan, lch, rut, phi, ty xng, ty) ring vo