49 (2) :272 276, 2003 A cta Zoologica S inica 3 (, 265200) (, 712100) 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 265200, Chi na) ( College of A ni mal Science and Technology, Northwestern A gricult ural U niversity, Yangli ng, S haanxi 712100, 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) : 272-276, 2003 ]. Key words Mouse, Early embryonic development, Gene expression, Protein, DD2PCR DD2PCR -,,, -,,,, (, 2002 ;, 2001 ;, 111 2001) ( 20 22 g),, 3 5 d,,,, 112, 11211, 20 5 IU/ PMSG, 48 h hcg 5 IU/, 10,,, ( Worad et al., 1997 ;, 11212 1996), 1 2001212213, 2002209225 3 (01S08) ( Y00023) [ This research was funded by Shandong Provincial Science Foundation (01S08) and Laiyang Agricultural College Foundation ( Y00023) ],, 42,, : E2mail : dkxbgs @lyac. edu. cn ν 2003 Acta Zoologica Sinica 1
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 01144 % 2 l RNasin (40 U/ l), 015 l 8 mmol/ L D TT, 2 Na 2 HPO 4 01024 % 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, 45 120 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, 70 10 min 2 11216 cdna Bassam et al. 11213 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 / 20 200, 200 l, ( Tris - ) 5 6 h, 40 U RNasin,, 3, DNA : PUC Marker ( Promega mrna ( < 012 ng) ) 11214 (RT) 11217 Oligo (dt) 20, PCR 50 10 100 ( 18 g), (019 %, 28 (Bauer et al., NaCl) 1, 15 l [ 10 1994),, 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 11218 SDS2PA GE 10 % 15 % (dt) 15 ( Promega ) 3 %, Tris2 11215 ( RT) cdna PCR (010268 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 )
74 2 49 11219 : Marshall (1984) 1 DD2PCR PAGE Fig11 DD2PCR amplicon PAGE map of mouse oocyte O 123 : (Muture oocyte) 2 211 P 2 5 10 3 Oligo (dt) 15, DD PCR, PA GE, DD PCR cdna ( 1) 212 P 1 P 2 P 3 5 10 3 Oligo (dt) 15, 1 2 4 8 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)
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 ), 312 2 10, 8, 2 mrna,, 5 8, 95 % 8 ; 8 3 ( 2), 90 %, ; 8 mrna, 5 %,,, 3 2 311, 1, ( / ) (De Sousa et al., (1 ), 1 1998) 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 2 313 mrna, 2 mrna 275
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