Cloning and sequencing of rat bcl-2 gene riched guanine and cytosine / 中国组织工程研究
Chinese Journal of Tissue Engineering Research
;
(53)2007.
Article
Dans Chinois
| WPRIM
| ID: wpr-592729
ABSTRACT
BACKGROUND:
Excessive apoptosis of ovary granulosa cell is a dominant cause of premature ovarian failure and bcl-2 gene is able to inhibit cell apoptosis. But studies demonstrate that,the guanine and cytosine (GC) content reaches 60% in the rat bcl-2 gene sequence. This gene cannot be amplified using routine polymerase chain reaction method.OBJECTIVE:
To clone and identify the bcl-2 gene riched GC. DESIGN,TIME ANDSETTING:
Open experiment was finished in the Laboratory of Medicine and Molecular Biology,Life Science School of Sun Yat-sen University from May to December in 2007. MATERIALS Wistar rats were purchased from Experimental Animal Center of Sun Yat-sen University. Ecoli DH5?was preserved by Laboratory of Medicine and Molecular Biology,Life Science School of Sun Yat-sen University; pMD18-T vector was purchased from Takara Biotechnology (Dalian) Co.,Ltd.METHODS:
The bcl-2-cDNA,in which GC accounted for 60.6%,was obtained by modified reverse transcription-polymerase chain reaction from kidney tissue of Wistar rats,and was cloned into vector-pMD18-T. Characterizations and sequencing of the pMD18-T-bcl-2 were carried out by polymerase chain reaction screening of individual bacterial colonies. MAIN OUTCOMEMEASURES:
Cloning and purification of bcl-2 gene cDNA; results after connecting bcl-2 gene cDNA to pMD18-T vector and transducting Ecoli DH5?,identification of positive clone and results of sequencing.RESULTS:
The bcl-2 gene was identified by the clone and DNA sequencing. DNA sequence analysis was consistent with Genebank sequence,with a 99% homology.CONCLUSION:
The gene riched GC is difficult to be amplified,bcl-2-cDNA can be cloned and constructed into cloning vector pMD18-T successfully by the efficient technique for other genes riched GC.
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
langue:
Chinois
Texte intégral:
Chinese Journal of Tissue Engineering Research
Année:
2007
Type:
Article
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