Cloning of Early Mouse Embryo Development Related Genes Using Modified DD RT2PCR

ISSN 100727626 CN 1123870ΠQ 2002 12 Chinese Journal of Biochemistry and Molecular Biology 18 (6) :728 732 DDRT2PCR 1) 2) 1) 2) 1) 1) 3 ( 1) 100080 ; 2) 100080) mrna (DDRT2PCR) DDRT2PCR M 22 42 50 Northern GenBank EST ed1 Northern ed1 22 M 42 mrna RT2PCR Northern RNA Q291 Q786 Cloning of Early Mouse Embryo Development Related Genes Using Modified DD RT2PCR LI Wen2yong 1) 2) YU Wei2dong 1) 2) LIU Gui2sheng 1) CHEN Qing2xuan 1) 3 ( 1) Institute of Genetics and Developmental Biology Chinese Academy of Sciences Beijing 100080 China ; 2) Graduate School of the Chinese Academy of Sciences Beijing 100080 China) Abstract The analysis of differential gene expression during preimplantation embryogenesis has been hindered by the paucity of biological material Improved mrna differential display RT2PCR method was used for the to2 tal RNA extraction from mouse M eggs two2cell embryos and four2cell embryos The starting material for each case was about fifty cells and the desired experimental results were obtained Seven differential amplicons were obtained and the most evident one was named ed1 which was recovered subcloned sequenced and identified by reverse Northern hybridization The BLAST programs were used to search NCBI2nr A fragment ed1 with high similarity to many sequences in the existing database was found but its full length and function are unknown Reverse Northern hybridization experiments indicated that ed1 was stage2specific expressive gene in the two2cell embryo and no blot in M eggs and four2cell embryo Key words mrna differential display RT2PCR reverse Northern hybridization early embryo development total RNA [10 ] 12 mrnas mrnas :2002204212 :2002206211 ( : G2000016107) [1 6 ] 3 :Tel : (010) 62553160 E2mail :qingxuanchen @yahoo com 1965 6 Received :April 12 2002 ; Accepted :June 12 2002 2amanitin Supported by National Basic Research Priority Program No G2000016107 3 Corresponding author Tel : (010) 62553160 E2mail :qingxuanchen @yahoo com [7 9 ] RNA

6 : DDRT2PCR 729 [3 11 22 ] RNase DNase2 RNA DNA (reverse genetics) Micro RNA Isolation Kit 120 l mrna 215 l (20 mgπml) - 20 20 l ( substractive hy2 bridization ) [12 ] mrna ( mrna differential display) [13 14 ] [20 21 ] DDRT2PCR [15 ] 914 l RNA 2 l (10 molπl) HT(15) C 68 8 min 40 10 min 816 l [4 l 5 RT 2 l DTT( 011 molπl) 1 l RNA [16 18 ] (40 UΠ l) 116 l dntp(250 molπl) 100 U Superscript 100 U M2MLV] 42 50 min 95 5 min 9 l 20 l DDRT2PCR DD2PCR[215 molπl 3 HT(15) C 015 molπl 5 HAP 1 2 molπl dntp 215 molπl MgCl 2 1XPCR 015 Ci [ 2 1 32 P ] datp 215U Taq DNA ] PCR :94 10 min 40 5 min 111 72 5 min ;94 30 s 40 2 min 72 90 s 11111 39 ;72 5 min 8 molπl 6 % - 20 2 d 1 1 2 Micro RNA Isolation Kit Stratagene 11214 Rneasy Mini Kit Qiagen DNA X X Labeling Kit TaKaRa 015 ml (PMSG) EP 100 l DEPC 10 (hcg) min 15 min 17 000 g 2 min Superscript Gibco RNase2free DNase M2MLV Taq DNA dntps p GEM2T (20 mgπml) - 20 X2Gal IPTG Promega [ 2 32 P ] 10 l DEPC 50 l ( datp [ 2 32 P] dctp ( : 111 BqΠmmol) 1 2 3 ) PCR PCR :94 10 min ; 3 HT(15) C : 5 2AA GC T T T T T T T T T T T T T T T T TC23 5 HAP1 : 5 2AAGCTTGATT2 min ;94 10 min ;94 1 min 42 90 s 72 90 s 20 GCC23 ;72 10 min PCR 115 % 112 PCR 2 1 2 1 PMSG hcg 6 1 2 5 PCR A B B 1 1 JM109 p GEM2T 1 3 ml LB A M 50 B 37 115 ml 22 25 42 15 PCR 115 ml GenBank [19 ] 1 2 3 PCR 6 % 8 l (3 molπl) 300 l 215 l 94 1 min 40 90 s 72 90 s 20 72 10 [20 ] [22 23 ] 1 2 2 RNA RNeasy Mini 11216 Northern Kit 30 l cdna DNA

730 18 Northern M ( ) 12 22 42 cdna RNA DNA pool 115 % 2 3 h 7 Hybond :1 %BSA 1 mmolπl EDTA 015 molπl 7 %SDS(pH 717) 65 10 min DNA 65 ed1 (early embryo development2related) : (015 %BSA 1 mmolπl EDTA 40 mmolπl ph 712 5 % SDS) 3 10 min ; (1 mmolπl EDTA 20mmolΠL ph 712 1 %SDS) 3 20 min PCR - 20 5 d 2 211 PCR DDRT2PCR Fig 1 2 ed1 212 PCR PCR Fig 2 : 2 3 4 6 7 5 T DNA 7 ed1 Fig 2 Recovered amplicons were reamplified by PCR and were resolved on 1 2 % agarose gel M:DNA marker ; 1 and 5 : no bands indicate unsuccessful clones ; 2 3 4 6 and 7 are successful clones ; 7 : ed1 fragment is indicated by arrowhead Fig 1 Portion of an autoradiogram of amplicons obtained from mrna differential display using M eggs and preimplantation mouse embryos 1 :Positive control ; 2 :Negative control ; 3 : 12cell embryo ; 4 :M 2 stage unfertilized eggs ; 5 : 22cell embryo ; 6 : 42cell embryo Specific amplicon ed1 is indicated by arrowhead 213 Northern Fig 3 ed1 385 BLAST GenBank NCBI2nr NCBI2est ed1 Northern Fig 4 : 3 : ed1 22 M 42 Fig 3 cdna sequence of ed1 fragment Underlined sequences indicate the primers used in reverse transcription and PCR

6 : DDRT2PCR 731 Fig 4 Results of reverse Northern hypridization (A) Result of 1 5 % agarose electrophoresis ; (B) Result of reverse Northern blot M:DNA marker ;1 : M 2stage unfertilized eggs ; 2 : 12cell embryo (no cdna pool smear band maybe something is wrong in RTor PCR ) ; 3 : 22cell embryo ; 4 :42cell embryo ;The blot represent result of hybridization of ed1 3 DDRT2PCR 30 1 RNA 0143 ng 1 22 0135 ng 42 cdna 0160 ng RNA RNA RNeasy Mini Kit DNA Micro RNA Isolation Kit 50 ( References) RNA Fig 1 RNA DNA Poly(A) + mr2 NA pg RNA rrna ed1 ed1 rrna rrna 50 M 50 22 42 3 HT(17) C 5 HAP1 PCR mrna cdna Superscript M2MLV DDRT cdna Northern DNA RT2PCR ed1 22 M 42 : ed1 22 mrna ed1 GenBank + EMBL + DDBJ + PDB EST 1 Nothias J Y Majumder S Depamphilis M L DePamphilis M L1 Regu2 lation of gene expression at the beginning of mammalian development J Biol Chem 1995 270 : 22077 22080 2 Schultz R M Regulation of zygotic gene activation in the mouse BioEssays 1993 15 : 531 538 3 Henery C C Miranda M Wiekowski M Wilmut I Depamphilis M L Repression of gene expression at the beginning of mouse development Dev Biol 1995 169 : 448 460 4 Ma J Svoboda P Schultz RM Stein P Regulation of zygotic gene acti2 vation in the preimplantation mouse embryo : global activation and re2 pression of gene expression Biol Reprod 2001 64 : 1713 1721 5 Aoki F Worrad D M Schultz R M Regulation of transcriptional activi2 ty during the first and second cell cycles in the preimplantation mouse embryo Dev Biol 1997 181 : 296 307 6 Latham K E Solter D Schultz R M Acquisition of a transcriptionally permissive state during the 12cell stage of Mouse embryogenesis Biol 1992 149 : 457 462 7 Simon A B Henrion G Renard J P Duranthon V Onset of zygotic transcription and maternal transcript legacy in the rabbit embryo Reprod Dev 2001 58 :127 136 8 Kelk D A Gartley C J King W A Incorporation of 3H2uridine into Dev Mol goat sheep and hybridembryos J Reprod Fertil Abstr Series 1994 13 : 120 128 9 Hoffert KA Anderson GB Wildt D E Roth TL Transition from ma2 ternal to embryonic control of development in IVMΠIVF domestic cat em2

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