본 연구에서는 쥐의 미성숙 난자의 체외 성숙과정에서 매우 특이적으로 발현되는 후보 단백질 변화를 동정하려는 목적으로 체외 성숙 과정에서 GV 단계의 미성숙 난자와 MIl 단계의 난자를 실험 시료로 사용하였다. 그리고 단백질 Chip은 선행 실험에서 가장 효과적인 CM10을 사용하여 SELDI -TOF MS 분석 장치를 이용하여 후보단백질을 동정하였다. MIl 단계의 미성숙 난자와 비교하여 GV 단계의 미성숙 난자에서 16개의 후보단백질이 높게 발현되었으며, 이때 발현된 후보 단백질 각각의 분자량은 8180(후보단백질 2개), 10226 (5개), 15767(5개), 16770(4개) 달톤(dalton)이였다. 또한 29개의 후보단백질은 MIl 단계의 미성숙 난자에서 높게 발현되었고 이들의 분자량은 각각 10832(3개)17743(8개)20122(3개)22131(3개) 24857(7개) 33507(5개) 달톤 이였다. 한편 전체 후보 단백질 45개의 분석을 Real time RT-PCR에서 수행하여 13개의 잠재적인 후보단백질을 확인 동정하였다.
본 연구에서는 쥐의 미성숙 난자의 체외 성숙과정에서 매우 특이적으로 발현되는 후보 단백질 변화를 동정하려는 목적으로 체외 성숙 과정에서 GV 단계의 미성숙 난자와 MIl 단계의 난자를 실험 시료로 사용하였다. 그리고 단백질 Chip은 선행 실험에서 가장 효과적인 CM10을 사용하여 SELDI -TOF MS 분석 장치를 이용하여 후보단백질을 동정하였다. MIl 단계의 미성숙 난자와 비교하여 GV 단계의 미성숙 난자에서 16개의 후보단백질이 높게 발현되었으며, 이때 발현된 후보 단백질 각각의 분자량은 8180(후보단백질 2개), 10226 (5개), 15767(5개), 16770(4개) 달톤(dalton)이였다. 또한 29개의 후보단백질은 MIl 단계의 미성숙 난자에서 높게 발현되었고 이들의 분자량은 각각 10832(3개)17743(8개)20122(3개)22131(3개) 24857(7개) 33507(5개) 달톤 이였다. 한편 전체 후보 단백질 45개의 분석을 Real time RT-PCR에서 수행하여 13개의 잠재적인 후보단백질을 확인 동정하였다.
The present study was conducted to gain insights into oocyte maturation and early embryo development, SELDI-TOF-MS was used to find the protein candidates that are specifically or prominently expressed in mouse oocytes at the in vitro matured metaphase II (MIl) and germinal vesicle (GV) stages. By u...
The present study was conducted to gain insights into oocyte maturation and early embryo development, SELDI-TOF-MS was used to find the protein candidates that are specifically or prominently expressed in mouse oocytes at the in vitro matured metaphase II (MIl) and germinal vesicle (GV) stages. By using selected CM10 chip, found 16 candidates which were up-regulated in GV stage oocytes compared with in MIl stage oocytes, molecular weight are 8180 (2 candidates), 10226 (5 candidates), 15767 (5 candidates) and 16770 (4 candidates) Da respectively. And 29 candidates were higher in MIl than in GV stage oocytes, molecular weight are 10832 (3 candidates), 17744(8 candidates), 20122 (3 candidates), 22131 (3 candidates), 24857 (7 candidates) and 33507 (5 candidates) Da, respectively. All (45) candidate (0.2 and 1.0 % error tolerances) were performed real time RT-PCR analysis and further selected 13 more potential candidates.
The present study was conducted to gain insights into oocyte maturation and early embryo development, SELDI-TOF-MS was used to find the protein candidates that are specifically or prominently expressed in mouse oocytes at the in vitro matured metaphase II (MIl) and germinal vesicle (GV) stages. By using selected CM10 chip, found 16 candidates which were up-regulated in GV stage oocytes compared with in MIl stage oocytes, molecular weight are 8180 (2 candidates), 10226 (5 candidates), 15767 (5 candidates) and 16770 (4 candidates) Da respectively. And 29 candidates were higher in MIl than in GV stage oocytes, molecular weight are 10832 (3 candidates), 17744(8 candidates), 20122 (3 candidates), 22131 (3 candidates), 24857 (7 candidates) and 33507 (5 candidates) Da, respectively. All (45) candidate (0.2 and 1.0 % error tolerances) were performed real time RT-PCR analysis and further selected 13 more potential candidates.
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제안 방법
Frozen-thawed GV and Mil stage oocytes were used to determine the expression of 14 genes during the oocyte maturation. The mRNAs from three sets of 50 GV or Mil stage oocytes were extracted by using the Dynabeads mRNA Direct Kit (Dynal Asa, Oslo, Norway) according to the manufacturer's instructions and converted into cDNA by reverse transcription of the RNA by using the Oligo (dT)12-18 primer and the Superscript reverse transcriptase enzyme (Invitrogen Co.
To determine the best condition for the discrimination between immature and mature oocyte, each four (n=4) from sample groups were analyzed on four ProteinChip (NP20, H4, CM10 and SAX 10) array and resulted in the selection of weak cation exchange ProteinChip Array (CM 10 ProteinChip Array) as the most effective ProteinChip Array.
대상 데이터
A potential candidate for the 24942 Da (upregulated in MII stage oocyte) is a casein kinase 2 (CK2) subunit beta. CK2 is present in the nucleolus, the site of ribosome biogenesis and copurifies with mammalian Pol I [7].
Preliminary identification through database searching highlighted 16 and 29 interesting potential candidates down and upregulated in Mil stage oocyte respectively. All (45) candidate (0.2 and 1.0 % error tolerances) were performed real time RT-PCR analysis and further selected 13 more potential candidates.The potential candidate of NADH- ubiquinone oxidoreductase was identified as a Mil stage specific protein.
성능/효과
type. Comparison of GV stage with Mil stage oocytes revealed 29 protein candidates were significantly highly expressed in Mil stage oocyte, at approximately 10831.79, 17743.78, 20122.1, 24857.48, 22131.43 and 33507.95 Da, and 16 protein candidates were highly expressed in GV stage oocytes, at approximately 8175.57, 15767.13, 10226.24 and 16770.37 Da.
참고문헌 (10)
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