Tap chi Cdng nghe Sinh ^pc H (1): 63-67, 2013
C A M U N G SU B I E U H I E N M O T S O G E N O A T I E M N A N G C U A N G U ^ EN B A O S O I PHOI C H U O T B A N G D j C H C H I E T T R U N G H E O G I A I £ ) O A N T U I MAM
Le T h i n h Long', Ngo Duy Binh^, Phan Kim Ngoc^, Hoang Nghia SOTI' ' Vi^n Sinh hgc nhiel dai, Vien Hdn tdm Khoa hgc vd Cdng nghe Viet Nam 'Dal hoc Khoa hpc lu nhien, Dai lioc Qudc gia, Thdnh phd Ho Chi Mmh
TOM T A T
Qua Irinh cam iing gen da tiem nang co the dugc lien hanh bang nhieu each khac nhau, Trong nghicn cim nay, chiing loi tien hanh cam iing sy bieu hien a miic phien ma mot so gen da tiem nang dac trung cho te bao goc phoi nhu oct4, nanog, rexl bang dich chief trimg heo giai doan tiii mam (germinal \esicle). Ket qua eho ihay, dich chiel tning heo co kha nang cam img sir bieu hien cua ocl4. nanog. rexl a mirc phien ma va su bieu hien ciia protein 0CT4 sau 3 tuan cam iing. Tuy nhien su bieu hien cua oct4. nanog trong te bao cam img yeu hem so vol te bao goc phoi khoang 1 4 lan con rexl bieu hien trong quan the te bao cam img rat \'eu Sau 3 tuan cam img chiing toi cung nhan thay su hinh thanh cac quan the te bao co hinh thai giong quan ihe te bao goc phoi (embryonic stem cell) ciing nhu quan the le bao goc cam ung (induced plunpotent stem cell) bang cac nhan lo xac dinh KSt qua Iren cho thay, d|ch chiei tning heo la mot nguon cung cap cac nhan to cam iing gen da tiem nang hieu qua va Hem nang
Tir khoa Dich chiel Irimg. gen da iiein ndng. c/udn the le bdo cam img, sir hieu hien gen, ldi ihiel ldp climmg irinh
MO DAU
Ciru Dolly duoc nhan ban thanh cong nam 1997(Wilmut e / a / , 1997) va chuot Cumulma duoc nhan ban thanh cong mot nam sau do (Wakayama el ai, 1998) da gop phan dat nen tang to lan cho lien trinh nghien ciru ve nhan ban vo linh ciJng nhu tai ihiel lap chuong Irinh ciia cac gen da tiem nang.
Te bao sinh duang duoc xir li bang cac tac nhan irc che qua Irinh methyl hoa nhu 5-aza cystidine va tie che enzyme khu nhom acetyl nhu Trichostatin A ciia histone 3 lam tang ti le phal tnen cua phoi bo den giai doan blastocyst (Enrighl et ai, 2003). Vice chuyen cac nhan to xac dinh nhu ocl4, c-inyc. soxl va klf4 vao nguyen bao soi phoi ehupl co kha nang tai ihiei lap chuang trinh bo gen tao thanh cac quan the te bao goc cam img da tiem nang co dac tinh giong le bao goc phoi (Takahashi and Yamanaka, 2006), Qua trinh xir ly phoi nhan ban vo tinh bang nhan to irc ch^ sy hoat dong ciia enzyme khii nhom acetyl ciia histone 3 giiip tang cucmg qua trinh tai ihiel lap bp gen ciia phoi nhan ban va lang Iy le ehupt nhan ban phat trien hoan chinh len 6 lan (Kyshigann et al. 2006) Hon nira, nguyen bao sai phoi ehupt CO kha nang hinh ihanh cac quan ihe le bao cam irng bieu hien gen da tiem nSng ihong qua
\icc cam ling bang dich chiel iriimg chuoi giai doan llii mam (Bui f / a / . 2008)
Tir nhiJng cong trinh tren, chiing loi lien hanh
nghien ciiu cam iing su bieit hien cac gen da tiem nang ciia nguyen bao soi phoi chuot bang djch chiel Iriing heo. Muc tieu cua nghien ciiru nay la khong nhiing tim ra nguon cam iing gen da tiem nang mm ma con giiip khac phuc nhugc diem lam bien doi di truyen bo gen trong te bao soma cua cac phucmg phap tren.
VAT LIEU VA PHUONG PHAP Thu dich chi^t trirng heo giai doan tiii mam
Buong trimg heo ihu nhan tir 16 mo sau do duoc dem ve phong thi nghiem Phirc hop trimg-cumulus se dugc thu nhan bang phuong phap choc hul Trimg giai doan tin mam (germinal \csicle (GV)) dugc loai bo cac te bao cumulus Mang zona pellucidae cua iriimg dugc loai bo bang dung dich acid lyrode Trimg loai bo mang zona pellucidae duoc u v6i 50 pi mdl trucmg HEPES (Chatol el al.
1990) chira ImM ATP, lOmM creline phosphate, 25 pg'ml creatine kinase va protein inhibitors (Sigma-Aldrich, My) Dich chiet Irirng GV dutrc chuan bi theo li le 1000 trirng trong KIO pi mni trucrng HEPES
Phu-ong phap cam iing ngu>(;n bao soi phiii chuot NiiuNcn bao sOi phoi chucit duoe tach hang l)25"crir\psin-EDTA (il>elone. Due) Sau do ducrc 6^
Le Thanh Long el ai
\u ly veil dung djch Hank chira 200 ng'ml Streptolysin O trong 40 phiit.Sau do te bao dugc u vai 200pl dung dich HEPES chiia dich chiet trung heo trong 40 phiit (Bui e! al, 2008). Te bao dugc xii ly voi dung dich HEPES khong chiia dich chiet tning heo dugc thyc hien lam doi chung Te bao duac xir ly va khong dugc xii ly dich chiet tning heo dugc nuoi trong moi truang DMEM 10% FBS ci 37"C, 5%
CO:,Qua trinh cam img duoe thyc hien trong 3 tuan luong duang voi 6 lan cay truyen. Sau khi nuoi cay, te bao duoc thu nhan de xac dinh su bieu hien mgt so dau chuan phan tu da nem nang bang phuang phap RT-PCR va nhuom mien dich te bao.
R T - P C R
mRNA long dugc thu nhan bang EasyBlue Kit (Intront. Han Quoc) va pha loang trong 20|il nuac DEPC (Sigma, My) Phan img RT-PCR duoc su dung de xac dinh sy bieu hien cac gen da tiem nang Trinh ty moi dugc sir dung nhu sau, moi cho ocl4 (GenBank Assession Number, NM_013633, moi XUOI 5'-GGCGTTCTCTTTGGAAAGGTGTTC-3', mOi nguoc 5"-CTCGAACCACATCCTTCTCT-3', cho san pham co kich thuac 312 bp), moi cho nanog (GenBank Assession Number, NM_028016,2, moi XUOI 5'-CTGCTCCGCTCC ATA ACT TC-3', mOi nguoc 5"- GCCCTCTTCTGGAGTGTCTG-3' cho san pham co kich thuoc 328 bp), moi cho rexl (moi XUOI 5'-ACG AGT GGC AGT TTC TTC TTG G G A - 3 ' . mill nguoc 5'-TAT GAC TCA CTT CCA GGG GGC ACT-3" cho san pham co kich thuac la 287 bp) (Takahashi. 2006), moi cho P-actin (GenBank Assession Number. NV1_007393.3, moi
\u6i 5"- GCTCTTTTCCAGCCTTCCTT-3', m6i ngugc 5"-TGATCCACATCTGCTGGAAG-3' cho san pham co kich thuoc la 295 bp)
Chu trinh nhiet 45"C Irong 30 phiit. 94"C trong 5 phiit; 94"C trong 45 giay, 55"C trong 60 giay. 72"C trong 60 giay. 34 chu ki. 72"C Irong 10 phiit Nhuom khiing the don dong
Te bao se dugc co dinh trong dung dich co dmh miiu (PBS chira 4% paraformaldehyde) Irong 20 phiit, Rua te bao hai lan viii dung dich rua (20mM Tns-HCL 0 9% NaCI. 0 0 5 % Tween 20) mdi \kn 10 phiit. Phu be mat te bao bang dung dich Blocking (PBS chira 4% Normal Goat Serum) trong 30 phiit U te bao \(Vi dung dich chira khang the khang 0 C T 4 f Santa Cruz Biotechnology. Santa Cruz, CA. USA) trong 1 h Sau dci Ie bao se dugc nia voi dung djch rua 3 lan moi lan 10 phiit Te bao dugc ii ven khang the thil cap Ale\a-Fluor-5!4-labeled chicken anti-
rabbit antibodies (Molecular Probes, My). Te bao dugc rira 3 ISn voi dung dich rira, moi lan 10 phuL T^ bao duoc quan sat duoi kinh hien vi huynh quang de quan sat su bieu hien cua 0 C T 4 ,
Dinh lirotig b a n phan sir bieu hien ciia cac gen da tiem nang
Sy bieu hien cua cac gen dugc dinh lugng tuong doi bang phan mem Image J (National Institute of Health. Bethesda. MD, My)
KET QUA VA BIEN LUAN
Djch chiet trirng cam sir bieu hi€n cac gen da tiem nang miJc phien ma
Dich chiet Irirng ctia ech (Xenopus) hay te bao goc phoi CO kha nang tai mo hinh hoa bg gen cua tS bao sinh duong thong qua qua trinh kich hoai gen da tiem nang nhu ocl4, nanog, sox2, rexl, c- mye, (Byrne el ai, 2003; Simonsson and Gurdon, 2004, Freberg el ai, 2007; Miyamoto el al, 2007) Trong nghien ciiu nay, chiing toi danh gia su bieu hien ciia mgt so gen da tiem nang (ocl4, nanog va rexl) cs mirc phien ma sau ba tuan cam irng bang dich chiet tning heo Sy bieu hien mirc phien ma cua cac gen ocl4, nanog vd rexl dien ra o tuan thii 3 (Hinh I A). Trong khi do cac nguyen bao sgi phoi chuot khong dugc cam ung voi dich chiet tning heo khong co sy bieu hien ciia gen oct4, nanog va rexl. Viec xii ly nguyen bao sgi phoi chuot voi Streptolysin O giiip tao cac 16 tren mang te bao chat cua nguyen bao sai phoi chuoi, do do cac nhan to tai thiet lap chuong trinh tu dich chiet trimg heo di vao te bao va hoat hoa gen da tiem nang trong te bao,
Ket qua phan tich bang phan mem ImageJ cho thay su bieu hien a miic phien ma cua oct4, nanog va rexl trong nguyen bao sgi phoi chuot cam iing bang dich chiet trimg heo yeu hon so vol su bieu hien cua chiing trong te bao goc phoi chuot (Hinh I) Su bieu hien ciia ocl4 va nanog a te bao goc phoi cao hon khoang 1.4 lan so v6i su bieu hien cua ocl't va nanog o quan the te bao cam iing, Su bieu hien cua gen rexl dien ra rat yeu, mirc do bieu hien ciia gen rexl trong te bao cam img yeu hon trong te bao goc phoi khoang 15 lan
Qua trinh cam img nay dugc thyc hien a hai doi tugng khac Ioai, dieu do cho thay cac nhan to cam img tir dich chiet trimg ciia dpng vat hifu nhu co linh linh dong va bao ton cao.
Tgp chl Cdngnghe Smh hpc 11(1): 63-67. 2013
Djch chiet tnj-ng heo cam sir bieu hicn gen odd mirc (ljch ma
Chiing toi ciing xac dudi su bicu hien a miic dich ma cua gen oct4 \i da> la gen da tiem nang dac . lrung nhat cho te bao goc phoi chuot Ket qua rdiuom iiuen dich te bao cho tliay mot so quan the nguyen
bao SOI phoi chuot cam irng bang dich cliiei trimg heo bieu hien prolein 0CT4 (Hinh 2) Sir bieu luen protem 0CT4 khong dien ra o tat ca cac quan llie lc bao cam inig.Trong khi 0CT4 o te bao goc phoi chuot bieu hien manh o tat ca quan the.Dieu do cho tliay, sir cam iiiig bieu luen prolein 0CT4 bang drch chiet trimg heo xa>' ra khong hoan toan
Hinh 1 A SL^ bigu hien ciia gen da ti§m nang trong te bao cam sa sanh s u bieu hien ciia cac gen bang ImageJ cho t h i y s u b i l u t l baogSc phoi
G e n e
Beracicnii Oct4 Sanog RexJ
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ang Irong le bao cam ung yeu hon trong
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^H 1
Hinh 2. Su b i l u hien protein 0 C T 4 A q u i n t h i te bao gee phoi, B, C quSn thS l i bao cam i>ng Protein 0 C T 4 6 l i bao g i c phoi b i i u hien manh (Al), irong khi dd cac quSn I h i te bao cam irng b i i u hien 0 C T 4 khong dong d i u ff cac quSn t h i ( B l , C l )
Su'hinh thanh cac quan thC'giong tebao got phoi Su hinh thanh cac quan ihc lc bao cam irng
giong lc bao goc phoi cho lha> \icc bicu hign
Quan Ihc tc bao cam irng giong lc bao gdc prolein 0CT4 dong \ai tro nil quan trgng trong
phoi CO su ph;il trien lii tc bao cam irng. co hinh qua Irinh l:ii Unci lap chuang Irinh 0CT4 la
d;ing iron deu, bt myl Iran nhan khac \cii cac cym prolein dac trirng cho te bao goc phoi. no tham gia
lc bao dirpc hinh thanh do sy lien k^l cac lc bao \ao \icc diau hoa kich hoat cac gen da licm ming
khac nhau khong phat iricn lir mpl lc bao ban dau kh:ic nhu nanog. so\I. foxd3 (?m\ and Thomson.
Le Thanh Long etai 2007), Ben canh do. sy bieu hien cua OCT4 irc
che sy hoat dgng ciia cac gen biet hoa n h u lamin (Bui el ai, 2008) bang each bam vao promoter ctia cac gen nay. Quan the te bao cam img chiing toi thu nhan dugc dien ra qua trinh thoai hoa sau 3 den 4 ngay co the do moi trucrng nuoi cay khong chiia mpt so nhan to can thiet cho qua Irinh nuoi cay te bao goc phoi. dac biet la lif (leukemia inhibitory factor), day la nhan to lie che qua trinh biel hoa ctia te bao goe phoi chuot ( H i n h 3 D 3 ) ,
Hinh thai quan the nguyen bao sai phoi chuot chimg toi ihu dugc giong nhu cac quan the te bao goc cam ling bang cac nhan to xac dinh (Takahashi.
Yamanaka, 2006), hay quan the te bao goc cam iing bang cac hoa chat (Kanatsu-Shinohara et ai, 2004), hay quan the te bao goc phoi cam img bang phuang phap dung hgp te bao sinh duong voi te bao goc phoi (Chad et ai, 2005), Hinh thai ciia quan the nguyen bao SOI cam iing va su bieu hien ciia 0 C T 4 cho thay quan the te bao chung toi thu dugc co sy bieu hien tinh da liein nang
%)
Hinh 3 Hinh thai quSn the le bao A, B. cum te bao hinh Ihanh do su' k i t cum cua nhieu t i bao khac nhau, hinh thai guar Ihe go g h i {mOi ten t r i n g hinh A) va tao ra khoang t r i n g giua cac t i bao (mui ten triing hinh B) C' quSn the t i bao g i c phoi phat triin tren nguyen bao soi phoi chuot bi bai hoat mitomycin C D l , D2, D3, s u hinh thanh, phat Inen va Ihoai hoa ciia quan the t i bao cam i>ng
KLT LU,-\N
Chiing Icii da thanh cong trong viec cam ung mot so gen da licm nang a inirc phien ma va djch ma bang dich chiet triing heo giai doan hii mam. IVlgt s6 quan ihe te bao cam irng dugc hinh thanh va bi6u hien proiein 0 C T 4 . day la mpl dac diem quan trpng de xac dinh tinh da tiem nang ciia tg bao g6c cam img hay le bao goc phoi Ket qua tren chimg to dich chiL'i tning heo la mot nguon cam img su bieu hien cac gen da Hem nang miii
TAi LIEU THAM KHAO
Bui HT. Wakayama S, Kishigami S, Kim JH, Van ThuLiii N. Wakayama T (2008) The cytoplasm of moii^c germinal vesicle stage oocytes can enhance sonialic cell nuclear reprogramming Devclopmenl
\35- 3')35-3y4.s
(. Iiad AC. Jiicclvii .A Douglas AM. Kevin E (2005) Niiclciii Reprogramming of Sonialic Cells After Fusion w iih I luman fc"mbr\onic Stem Cells, .Science 309:
Chatol CL, Lewis JL, Ton-es 1. Ziomek CA (1990) Developmenl of 1-cell embryos from different strains of mice in CZB medium, Biol Reprod 42- 432-440 Enright BP. Kubota C, Yang X, Tian XC (2003) Epigenetic characteristics of donor cells treated by trichostatin A or 5-Aza-2'-deoxycytidme and developmenl of embryos cloned from treated donor colls Biol Reprod 69:896-901,
Freberg CT, Dahl JA, Timoskainen S, Collas P (2007) Epigenetic reprogramming of 0CT4 and NANOG regulatory regions by embryonal carcinoma eel! exUacL Mot Biol Cell 18: 1543-1553.
Kanatsu-Shmohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S. Kalo T, Kazuki Y.
Toyoshima M, Niwa O, Oshimura M, Heikc T, Nakahata T. and Shinohara T (2004) Generation of pluripoleni stem cells from neonatal mouse testis. Cell 119:1001-1012,
Kishigami S. Mizulani E. Ohta H. Hikichi T, Thuan NV. Wakayama S. Bui HT. Wakayama T (2006) Significant improvement of mouse cloning technique by Ireatment with Uichostalin A after somatic nuclear transfer Biochem Biophys Res Commun 340 183-189.
Tap chi Cong nghe Sinh hpc 11(1): 63-67, 2013
Miyamoto K, Furusawa T, Ohnuki M, Goel S. Tokunaga Takahashi K. Yamanaka S (2006) Induction of T, Minami N, Yamada M, Ohsumi K. Imai H (2007) PluripotenI Stem Cells from Mouse Embryonic and Reprogramming events of mammalian somatic cells Adult Fibroblast Cultures by Defined Factors. Ct'/n 26 induced by Xenopus laevis egg extracts. Mot Reprod Dev 663-676
74. 1268-1277-
Wakayama T, Perry A C, Zuccotti M, Johnson K R.
Pan G, Thomson JA (2007) Nanog and transcnpUonal Yanagimachi R (1998) Full-term development of mice networks m embryonic stem cell pluripolency Cell from enucleated oocytes injected with cumulus cell nuclei.
Research \1 42-49 Nature 394 369-374
Simonsson S, Gurdon J (2004) DNA demethylation is Wilmul L Schmeke AE. VlcWhir J, Kind AJ. Campbell necessary forlheepigeneUc reprogramming of somatic cell KH (2000) Viable offspring denved (rom fetal and adiili nuc\ei Nat Cell Biol 6 984-990 mammalian cells .\ai Genet 24 372-376
I N D U C I N G P L U R I P O T E N T G E N E E X P R E S S I O N O F M O U S E E M B R Y O N I C F I B R O B L A S T B Y P O R C I N E G E R M I N A L V E S I C L E O O C ^ T E E \ T R . \ C T
Le Thanh Long'*, .Ngo Duy Binh^ Phan Kim Ngoc", Hoang Nghia Son' Inslilule of Tropical Biology, Vietnam Academy of Science and Teclinolog}
'University of Science, Ho Chi Mmh City
SUMMARY
Mammalian plunpotent genes can be induced by many ways. In this study, we induced pluripotnpent genes of mouse embryonic fibroblast such as o<.i4, nanog, rexl using germinal vesicle porcine oocyies extract Tltc results showed Ihat germinal vesicle porcine oocytes cxtnicl can induce the transcnpt expression at oii4, nanog, and rexl However, ocl4 and nanog expression in mouse embiyonic fibroblast induced by gemunal vesicle porcine oocytes extract was lower Ihan mouse cmbryomi. stem cells The rc.\l expression was very lov\ in mouse
n bryo fib oblast induced by gemunal vesicle poicinc ooctytes extract Protein 0CT4 was expressed after due nfe, se en bryonic fibroblast 3 weeks A lew of induced colonies expressed OCT4 protein 0CT4 protein ex| e s o duced colonies was also lower than cnibrj'onic stem cells Induced colonies vvere fonned alter 3 eek nducn th procme germinal vesicle oocyte extract and matained their embryonic stem like colony norpi ology tro 3 to 4 days, then induced colony morphology was degraded and changed iheir morphology Cell lon e nduced by genninal vesicle poicine oocytes extract have the same morphology lo mouse cmbrvomc s n ell o ndu ed plunpotent stem cells denved from defined faclors The\ had smooth shape and round bou dary 1 d iguished from other cell clusters in rough shape jnd no boundary hnevKhich iire \er\ miponanl o de en embryon c stem as colony morphology Thus, germinal \esicle porcine oocytes extract ina> be useful lo supply inducing pluripoleni genes factors of mouse embryonic libroblast and we also found some induied colonies were formed look like induced plunpolenl slem cell den\ed from detined factor emphasi/iny ooc>le extract has great potency for inducing induced plunpoieni sieni cells
Keywords gene expression, iniliucd plunpoieni siem cell cotonx oocMc exiracl, pluripoleni gene repi vgram m ing.
Author for correspondence E-mail lelimgI51 Iha amail c o
