선택한 단어 수는 입니다.
최소 단어 이상 선택하여야 합니다.
최소 단어 이상 선택하여야 합니다.
선택한 단어 수는 30입니다.
최대 10 단어까지만 선택 가능합니다.
최대 10 단어까지만 선택 가능합니다.
제안 방법
- The metaphase chromosomes were treated with RNase A (100 mg/mL in 2X SSC) for 1 h at 37℃. After rinsing three times with 2X SSC, the slides were dehydrated in a series of ethanol solutions of 70%, 85%, and 100% for 5 min, respectively. The chromosomes were then denatured in a 70% formamide solution for 2 min and dehydrated in another series of ethanol washings.
- In order to evaluate the colinearity between the am-plified gene copy number and the scu-PA expression, the DNA, RNA, and protein quantity in the scu-PA was examined. First, to characterize the genetic structure and copy number of the scu-PA gene inserted into and amplified in the genome of the MGpUK cell lines, a genomic Southern analysis was performed on the finally selected MGpUK-5 cell line (Fig.
- The scu-PA gene was obtained by a PCR (polymerase chain reaction) of the genomic DNA isolated from SVpUK. The PCR was performed using the sense primer 5WCCAAGCTTG CCACCATGAGAGCCCTG- CTGGCGCGCCTG-3' with a Hindlll enzyme site and antisense primer 5r-TCCCCCGGGTCAGAGGGCCAG- GCCATT-3' with a Smal enzyme site. The PCR products of the scu-PA gene; which were about 1.
- The influence of the gene amplification on transcription and translation was analyzed by comparing the relationship between the amplified gene copy numbers, the transcript amount, and the secreted protein of the MGpUK-5 cell line (Table 1). With gene amplification up to the stage of 2.
- In order to examine the location of the scu-PA gene, the metaphase chromosome of the MGpUK-5 cell line was hybridized with a biotin-labeled scu-PA specific probe and treated with fluorescein avidin. The slides were mounted with PI and DAPI, and then analyzed 쵸nd photographed. The figure shows the photographs stained with (a) DAPI and (b) fluorescein avidin, respectively.
- To detect the location of the amplified scu-PA genes in the chromosome; in situ hybridization was performed on the genomic DNA of the MGpUK-5 cell line using a biotin-labeled specific scu-PA gene probe (Fig. 8). Most of the amplified scu-PA genes were located within one chromosome, yet 5-10% of them were also found in other chromosomes including the previous one.
- The influence of the gene amplification on transcription and translation was analyzed by comparing the relationship between the amplified gene copy numbers, the transcript amount, and the secreted protein of the MGpUK-5 cell line (Table 1). With gene amplification up to the stage of 2.0 μM of MTX; the scu-PA gene was amplified approximately 150-fold, its transcripts 45-fold, and the scu-PA protein 70-fold; based on two independent experiments. The gene amplification was three and two times higher than the RNA and protein increment, respectively.
대상 데이터
- A DHFR-deficient mutant of CHO cell line, DG44 [49], was kindly donated by Professor Chasin (Columbia University; USA). The cell line was sub-cultivated in MEM-a (GibcoBRL, Grand Island, NY, USA) containing nucleosides and deoxynucleosides supplemented with 10% FBS (GibcoBRL) at 37℃ in a 5% CO2 air atmosphere.
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