Yamagiwa Hiroshi, Bolander Mark E, Sarkar Gobinda
Dept of Orthopedic Surgery, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA.
Mol Biotechnol. 2004 Feb;26(2):111-6. doi: 10.1385/MB:26:2:111.
We tested three different standard ligation conditions (37 degrees C for 30 min, 16 degrees C for 24 h, and 4 degrees C for 48 h) to generate dumbbell-shaped oligonucleotides (ODNs) as transcription factor decoys for SOX9 and alphaA-crystallin binding protein 1 (CRYBP1), which are positive and negative transcriptional regulators for type II collagen expression in chondrocytes. Decoy ODN for CRYBP1 was successfully produced as a "dumbbell" by all three conditions. A small amount of decoy ODN for SOX9, however, remained unligated under all three ligation conditions. Ligation at 4 degrees C for 48 h appeared to be the least desirable for SOX9 ODN. Transfection experiments with the SOX9 ODN ligated in different conditions and a luciferase-based reporter system also supports this conclusion. In general, shorter incubation time produced more acceptable results for this ODN than incubation for a longer time. These data suggest that different ligation conditions should be tested prior to creating dumbbell-shaped ODNs for transfection experiments.
我们测试了三种不同的标准连接条件（37℃ 30分钟、16℃ 24小时和4℃ 48小时），以生成哑铃状寡核苷酸（ODN）作为SOX9和αA-晶状体蛋白结合蛋白1（CRYBP1）的转录因子诱饵，它们分别是软骨细胞中II型胶原蛋白表达的正性和负性转录调节因子。在所有三种条件下，CRYBP1的诱饵ODN均成功制备成“哑铃”状。然而，在所有三种连接条件下，仍有少量SOX9的诱饵ODN未连接。对于SOX9 ODN，4℃ 48小时的连接似乎是最不理想的。用在不同条件下连接的SOX9 ODN和基于荧光素酶的报告系统进行的转染实验也支持这一结论。一般来说，较短的孵育时间比长时间孵育能产生更可接受的结果。这些数据表明，在制备用于转染实验的哑铃状ODN之前，应测试不同的连接条件。
