Gene expression of mouse in early embryonic development 3

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1 49 (2) : , 2003 A cta Zoologica S inica 3 (, ) (, ) Gene expression of mouse in early embryonic development 3 ZHU Xin2Chan ZHAN G Yong WAN G Bao2Wei ( Depart ment of A ni mal Science, L aiyang A gricult ural College, L aiyang, S handong , Chi na) ( College of A ni mal Science and Technology, Northwestern A gricult ural U niversity, Yangli ng, S haanxi , Chi na) Abstract A modification of the mrna differential display method was used to analyze differential gene expression during early embryonic development in the mouse. This method detects appropriate changes in the temporal pattern of expression of amplicons and proteins by DD2PCR and SDS2PA GE analysis. In addition, it indicates that the activity of genes during early embryonic development was temporal ; different degrees of activation expression translation occurring with chang2 ing spatio2temporal and environmental conditions. The selectivity of gene expression and levels of transcription and transla2 tion are a key controlled2regulated position during early embryonic development in the mouse [ Acta Zoologica Sinica 49 (2) : , 2003 ]. Key words Mouse, Early embryonic development, Gene expression, Protein, DD2PCR DD2PCR -,,, -,,,, (, 2002 ;, 2001 ;, ) ( g),, 3 5 d,,,, 112, 11211, 20 5 IU/ PMSG, 48 h hcg 5 IU/, 10,,, ( Worad et al., 1997 ;, ), , (01S08) ( Y00023) [ This research was funded by Shandong Provincial Science Foundation (01S08) and Laiyang Agricultural College Foundation ( Y00023) ],, 42,, : E2mail : edu. cn ν 2003 Acta Zoologica Sinica 1

2 2 : 1 Table 1 Collection of mouse embryonic cell during early development 273 Early development stage Mature oocyte Fertilizedegg First cleavage Second cleavage Third cleavage Morula Preblastula hcg 0 ( Injected hcg starting time 0 h) 15 h 28 h 44 h 55 h 65 h 75 h 85 h (M ) (Collected embryo cell) (Oocyt) 1 (12cell) 2 (22cell) 4 (42cell) 8 (82cell) (Morula) ( Preblastula),, mmol/ L KCl, 50 mmol/ L D TT p H 815), 2 l 10, PBSS mmol/ L dn TP ( da TP d GTP dctp dttp), [ PBS ( 018 % NaCl 0102 % KCl % 2 l RNasin (40 U/ l), 015 l 8 mmol/ L D TT, 2 Na 2 HPO % KH 2 PO 4 p H 714 DEPC l AMV2RT (20 U/ l), 815 l ddh 2 O (RNase2 H 2 O ), 1 %NCS - ] free), 20 l, min ;,, PBSS 3,,,,, 1 mg/ ml, p H 813), 3 l 2 mmol/ L dn TP ( da TP d GTP, ( M dctp dttp), 1 l (6215 mol/ L ), 1115 l ddh ), 2 O, 94 3 min, 015 l Taq DNA (1 8 ) PBSS min 50 1 min 70 2 min ; 38 : 94 ( ), 1 min 50 1 min 70 2 min ; 1, PBSS 3, min cdna Bassam et al mrna Boehringer PCR : 4 l 10 PCR ( 100 mmol/ L Tris2HCl, 15 mmol/ L MgCl 2, 500 mmol/ L KCl, 30 l ; : 94 3 (1991), PA GE ( 5 % 7 %, 10 % 20 %), 30 Mannheim mrna Capture kit, cm, 013 mm, 6 8 l / , 200 l, ( Tris - ) 5 6 h, 40 U RNasin,, 3, DNA : PUC Marker ( Promega mrna ( < 012 ng) ) (RT) Oligo (dt) 20, PCR ( 18 g), (019 %, 28 (Bauer et al., NaCl) 1, 15 l [ ),, mm Tris2HCl (p H 618), 2 % -, 1 % RT2PCR P240 ], 100 % : P 1. GA TCAA GTCC P 2. GTTTTCGCA G 2 min P 3. GA TGACTGGC Oligo SDS2PA GE 10 % 15 % (dt) 15 ( Promega ) 3 %, Tris ( RT) cdna PCR ( M Tris 012 M 011 % SDS p H Bauer (1994), RT2PCR 813), 10 l,, 25 ma RT : 5 l 5xRT 5 6 h ( (250 mmol/ L Tris2HCl, 40 mmol/ L MgCl 2, 150 )

3 : Marshall (1984) 1 DD2PCR PAGE Fig11 DD2PCR amplicon PAGE map of mouse oocyte O 123 : (Muture oocyte) P Oligo (dt) 15, DD PCR, PA GE, DD PCR cdna ( 1) 212 P 1 P 2 P Oligo (dt) 15, DD2PCR, PA GE ( 2), 213 SDS2PAGE SDS2PA GE 2 DD2PCR PAGE Fig12 DD2PCR amplicon PAGE map during early embryonic development O : (Mature oocyte) E 1 : 1 (12cell stage) E 2 : 2 (22cell stage) E 3 : 4 (42 cell stage) E 4 : 8 (82cell stage) E 5 : (Morula) E 6 : ( Preblastula) Marker : (Molecular weight marker PUC) A1 6 : 1 ( Product of primer 1 amplification) B1 5 : 2 ( Product of primer 2 amplification) C1 5 : 3 ( Product of primer 3 amplification)

4 2 : 3 SDS2PAGE Fig13 Protein SDS2PAGE pattern during early embryonic development O : ( Mature oocyte) E 1 : 1 ( 12cell stage) E 2 : 2 ( 22cell stage) E 3 : 4 ( 42cell stage) E 4 : 8 (82cell stage) E 5 : ( Morula) E 6 : ( Preblastula) Marker : ( Molecular weight of marker protein) 1 11 τ : ( Protein com2 ponent) ( 3), (A 4 A 5 A 6 B 3 B 4 B 5 C 4 C 5 C 6 ), , 8, 2 mrna,, 5 8, 95 % 8 ; 8 3 ( 2), 90 %, ; 8 mrna, 5 %,,, , 1, ( / ) (De Sousa et al., (1 ), ) 8, / 4 8 ( A 1 C 1 ), mrna,, mrna 2, ( 2),, 8 DNA, (A 3 B 1 C 2 ), mrna (A 2 B 2 C 3 ),,,,, 8 A 4 B 2 C 4, A 5 B 4 C 5, A 6 B 5 C 6, 8, (12 ), 2 2 ( 2, 3), 2, 2 (, 1997 ; Couge et al., 1998), ( / ),,,,, ( A 2 B 2 C 3 ),,, ( A 3 B 1 C 1 C 2 A 1 ),, mrna mrna, 2 mrna 275

5 ,, mrna (Lat ham et al., 1991), mrna,, mrna,,,, mrna,,,, , 1, , 3, 10 ( Wang et al., 1998) [,,, 1997, 2. mrna, 18 (1) : ] mrna ( 2, 3) ( References) Bauer, D., P. Warthoe, M. Rohde and M. Strauss 1994 Detec2 tion and differential display of expressed genes by DDRT2PCR. Methods and A pplication 4 : S97 S108. Bassam, B. J., G. Caetano2Anolles and P. M. Gresshoff 1991 Fast and sensitive silver staining of DNA in polyacrylamide gels. A nalytical Biochemist ry 196 : Couge, R. C., P. Marshourn, B. E. Gordon, W. Nunley and Y. M. Huet2Hudson 1998 Nitric oxide as a regulator of embryonic development. Biology of Reproduction 58 : De Sousa, P. A., A. Caveny, M. E. Westhusin and A. J. Watson 1998 Temporal patterns of embryonic gene expression and their dependence factors. Theriogenology 49 : Fan, H. Y and Z. M. Yang 2001 Effects of ethanol on the development of mouse preimplantation embryosin in vit ro. Acta Zool. Si n. 47 (1) : [, (1) : ] Hu, J., Y. Q. Zhang, X. P. Liu, R. A. Wang and J. R. Xu 2001 Expression of Smads proteins in rat testis during postnatal development. Acta Zool. Si n. 47 (5) : [, 2001 Smads. 47 (5) : ] Latham, K. E., D. Solter and R. M. Schultz 1991 Activation of a two2cell stage2specific gene following transfer of heterologous nu2 clei into enucleated mouse embroyos. Mol. Reprod. Dev. 30 : Li, Y. P., H. Shen, Z. Gu and J. K. Zuo 1997 A prelimininary study on the dviductal cytokines in controlling the development of mammalian early embryos. Zoological Research 18 (1) : Marshall, T Detection of protein in polyacrylamide gel using an improved silver strain A nalytical Biochemist ry 136 : 340 Wang, C. C. and C. L. Tsou 1998 Post2genome study pro2 teomics. Acta Biochem. Biophy. Si n. 30 : Worad, D. M. and R. M. Schultz 1997 Regulation of gene ex2 pression in the preimplantation mouse embryo temporaland spatial patterns of expression of the transcription factor SP1. Mol. Re2 prod. Dev. 46 : Yan, Y. Q., J. H. Tan and L. X. Zhang 2002 Expression of TGF2 and EGF2R mrna in the early embryonic development of mice. Acta Zool. Si n. 48 (4) : [,, 2002 TGF2 EGF2R mrna. 48 (4) : ] Zeng, W. Q. and F. Z. Sun 1996 Using gene trap screen to in2 dentify developmentally regulated genes in mammals. Prog. Biochem. Biophys. 23 (3) : [, (3) : ]

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