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제안 방법
- Recently, an efficient transgenic production system was established using piggyBac transposase and transposon elements[2,3]. In this study, we examined cumate-inducible transgene expression and loxP element-mediated transgene recombination using Cre recombinase in avian cells.
- In birds, however, there are relatively few reports on the application of recombinase-mediated gene cassette exchange using the chicken DT40 cell line [13,15]. In this study, we examined the cumateinducible transgene expression system and loxP element-mediated transgene recombination using Cre recombinase in avian cell lines. These versatile technical platforms could be efficiently adapted for use in functional genomics studies and for the generation of transgenic poultry to regulate the expression of specific target genes.
- 5 mM), 1 unit Taq DNA polymerase, and 10 pmol forward and reverse primer (Table 1). Quantitative RT-PCR analysis was performed using the iCycler iQ Realtime PCR detection system (Bio-Rad, Hercules, CA, USA) and EvaGreen (Biotium, Fremont, CA, USA). The PCR parameters were as follows: an initial incubation at 94℃ for 5 min, followed by 35 cycles at 94℃ for 30 s, 60℃ for 30 s, and 72℃ for 30 s.
- To examine the dose-dependent control of GFP transgene expression by cumate, flow cytometry was performed following induction with different concentrations of cumate. In the cumate- GFP cells, the percentage of GFP-expressing cells increased continually (Figure 2).
- One day after lipofection, 10 μg puromycin/mL was added to select the sublines stably transfected with the transgene. To induce transgene expression, the cumate solution (System Biosciences, USA) was added, and the culture media were changed every 3 days. For the translocation experiments, the loxP66-eGFP-loxP71 vector was co-transfected with Cre recombinase using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol.
대상 데이터
- 05% trypsin-ethylenediaminetetraacetic acid, the cells were resuspended in PBS containing 1% BSA and strained through a 40 μm cell strainer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for flow cytometry. Data analysis was performed using FlowJo software (Tree Star Inc., Ashland, OR, USA; http://www.treestar.com).
데이터처리
- 3 software (SAS Institute, Cary, NC, USA). Significant differences between the groups were determined using the general linear model (PROC-GLM) in SAS software. Differences between treatments were deemed significant when the p value was less than 0.
- Statistical analysis was performed using the Student’s t-test in SAS version 9.3 software (SAS Institute, Cary, NC, USA).
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