работа «Биологические компьютеры» (стр. 7 из 9)
Приложение БПРИЛОЖЕНИЯ РАЗРАБОТЧИКОВ
Молекулы siRNA (Язык оригинала).
The sequences of the ribo-oligonucleotides are given in Supplementary Table 1. T1 and T2 siRNAs were annealed from gel-purified oligomers (Proligo); gel-purified SI4 siRNA (Proligo) was used as provided by the manufacturers. FF3 and FF4 siRNAs were annealed from desalted oligomers (Dharmacon) after purification from 20% denaturing PAGE. To anneal RNA oligomers, equimolar amounts (50 or 200
M) of the sense and antisense oligomers were mixed in 50 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA and 0.5 U/ l Superase-In (Ambion), heated to 95 °C and slowly cooled down to 10 °C in a PCR block for 50 min. The size and purity of the siRNAs were verified using 3.5% Metaphor agarose gel (Cambrex).Молекулы siRNA (Технический перевод)
Последовательности рибо-оглинуклеотидов приведены в дополнительной таблице 1. Т1 и Т2 cиРНКотжигаются из гелевых очищенных олигомеров(Proligo). FF3 и FF4 сиРНА – из опресненных олигомеров (Dharmacon). Для производства олигомеров РНК, еквимолярные количества (50 или 200 моль) олигомеров были смешаны в in 50 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA и 0.5 U/
l Superase-In (Ambion), нагреты до 95 °C и медленно охлаждены до 10 °C в течении 50 мин. Размер и чистота сиРНК подтверждены, используя 3.5% агарозный гель (Cambrex) Клеточные культуры (Язык оригинала).
Serum-free media (SFM) adapted 293-H cells (Invitrogen) were used throughout the experiments. The cells were grown at 37 °C, 100% humidity and 5% CO2. The cells were initially transferred into CD-293 medium and a week later moved to the Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 0.1 mM of MEM nonessential amino acids (Invitrogen), 0.045 units/ml of penicillin and 0.045
g/ml streptomycin (penicillin-streptomycin liquid, Invitrogen), and 10% FBS (Invitrogen). The adherent culture was maintained indefinitely in this medium by trypsinizing with trypsin-EDTA (0.25% trypsin with EDTA 
Na4, Invitrogen) and diluting in a fresh medium upon reaching 50–90% confluence. For transfection experiments, 
90–120 thousand cells in 1 ml of complete medium were plated into each well of 12-well uncoated glass-bottom (MatTek) or plastic (Falcon) plates and grown for 24 h. Shortly before transfection, the medium was replaced with 1 ml DMEM without supplements with a single medium wash step. Transfection mixtures were prepared by mixing all nucleic acids, including the plasmids and the siRNAs into 40 l of DMEM. 2.4 l of the Plus reagent (Invitrogen) was added to the final mix and incubated for 20 min at 24 °C. In parallel, 1.6 l lipofectamine (Invitrogen) were mixed with 40 l DMEM. Plus- and lipofectamine-containing solutions were mixed and incubated for 20 more min at 24 °C before application to the cells. The transfection mixture (typically 90 l) was applied to the wells and mixed with the medium by gentle shaking. Three hours after transfection, 120 l FBS was added to the wells and the cells were incubated for up to 48 h before the analysis.The cells were prepared for the fluorescent-activated cell sorting (FACS) analysis by trypsinizing each well with 0.5 ml 0.25% trypsin-EDTA, collecting the cell suspension and centrifuging at 2,655 gs for 2 min. Trypsin was removed and the pellet resuspended by short vortexing in 0.5 ml PBS buffer (Invitrogen).
Клеточные культуры (Технический перевод)Медиатор без сыворотки (SFM) и приспособленный клетки 293-Н (Invitrogen) были использованы в экспериментах. Клетки выращивались при температуре 37 °C, 100% влажности и 5% CO. К клеткам были впоследствии добавлены 0.1 mM несущественных аминокислот, 0.045 единиц\моль пенициллина, 0.045
г/моль стрептомицина и 10% FBS.Для экспериментов с трансфекцией приблизительно 90-120 тысяч клеток в 1 мл определенной среды выращивались примерно 24 часа. Незадолго до трансфекции, среда заменялась 1 мл DMEM без добавок. Смеси для трансфекции были изготовлены, смешивая все нуклеокислоты, включая плазмиды и сиРНК, с 40
l DMEM. Через 3 часа после трансфекции, добавлялось 120 l FBS, клетки инкубировались до 48 часов перед анализом.Использование микроскопа (Язык оригинала)
All microscope images were taken from live cells grown in glass-bottom wells in the transfection medium supplemented with 10% FBS. We used the Zeiss Axiovert 200 microscope equipped with Sutter filter wheels, Prior mechanized stage and an environmental chamber (Solent) held at 37 °C during measurements. The images were collected by an Orca ERII camera cooled to -60 °C, in the high precision (14 bit) mode using a 20
PlanApochromat NA 0.8, PH2 objective. The collection setting for the fluorophores in crosstalk measurements and DNF evaluation experiments were S500/20x (excitation) and S535/30m (emission) filters for ZsYellow; and S430/25x (excitation) and S470/30m (emission) for AmCyan. A dichroic mirror 86004v2bs (Chroma) was used for both fluorophores. In CNF evaluation experiments the settings were: S565/55x (excitation) and S650/75m (emission) filters with a dichroic mirror 86021bs (Chroma) for dsRed- monomer, and AmCyan settings as above. For the anticorrelated output experiment we used yellow fluorescent setting as above and S565/25x (excitation) and S650/75m (emission) filters with a dichroic mirror 86007bs (Chroma) for dsRed-monomer. Data collection and processing were performed by the Metamorph 7.0 software (Molecular Devices). After background subtraction, the relative intensities of the internal transfection control and the reporter protein were adjusted to equalize the apparent intensity of both in the negative control experiments. The settings were applied uniformly to all images taken from the crosstalk experiments and DNF evaluations. A different setting was applied to the images taken from the CNF evaluation experiments and to the anticorrelated output experiments because the constructs had a different baseline fluorescence.Использование микроскопа (Технический перевод)
Все образы из микроскопа были взяты, используя живые клетки. Мы использовали микроскопы Zeiss Axiovert 200. Образы были отсняты фотоаппаратом Orca ERII, охлажденной до -60 °C, с большой точностью (14 бит), используя объектив 20
PlanApochromat NA 0.8, PH2. Для ДНФ испоьзовались такие фильтры: S500/20x (возбуждение) и S535/30m (подавление) для ZsYellow; S430/25x (возбуждение) и S470/30m (подавление) для AmCyan. Для КНФ: фильтры S565/55x (возбуждение) и S650/75m (подавление) для dsRed-monomer; и такие же для AmCyan. Измерение и анализ информации ( Язык оригинала)In crosstalk measurements and DNF evaluation experiments the cells were analyzed on a BD LSRII flow analyzer. ZsYellow was measured using a 488 nm laser and a 530/30 emission filter. AmCyan was measured with a 405 nm laser and a 450/50 emission filter. The data were analyzed using FlowJo software (FlowJo LLC). For each sample, the normalized ZsYellow level was calculated by dividing the compensated mean ZsYellow intensity in AmCyan-positive cells (obtained by putting the threshold at the highest autofluorescence value of AmCyan-negative cells) by the compensated mean AmCyan value in these cells. The normalized values collected in a single experiment were further divided by the ZsYellow/AmCyan ratio in the negative control samples. In the DNF evaluation experiments, the ratios were normalized to the lowest value in the unrepressed group of experiments. The same analyzer was used for the CNF evaluation experiments with LacI repressor. AmCyan was measured with a 405 nm laser and a 525/50 emission filter and dsRed-monomer with a 488 nm laser and a 575/26 emission filter. For each sample, the normalized dsRed level was calculated by dividing the compensated mean dsRed intensity (corrected for the cell autofluorescence) in AmCyan positive cells by the compensated and corrected mean AmCyan values in these cells. These values were divided by similarly calculated values in the negative controls where nonsense siRNA was applied at the same concentration as in the experiments. A MoFlo cell sorter (Darko) was used to analyze the C1 evaluation with LacI-KRAB repressor and the anticorrelated output experiments. ZsYellow was measured with a 488 nm laser and a 530/40 emission filter and dsRed monomer with a 568 nm laser and a 630/30 emission filter. The data from C1 evaluation were processed similarly to those described above. In the anticorrelated output experiment, for each reporter the mean value of its intensity in the transfected cells was weighted by the relative number of transfected cells and these numbers were independently factored for
each reporter to the higher of the two values. All reported values (except for the data in Supplementary Fig. 3) are averages of two independent experiments, and at least 30,000 qualified events were collected for each sample.Измерение и анализ информации (Технический перевод)
Для измерение уровня помех и оценивания ДНФ клетки анализировались на BD LSRII анализаторе. ZsYellow измерялся, используя 488 нм лазер и 530/30 эмиссионный фильтр. AmCyan измерялся 405 нм лазером и 450/50 эмиссионным фильтром. Информация измерялась программным обеспечением FlowJo (FlowJo LLC). Для каждого образца нормализованный уровень ZsYellow был рассчитан делением компенсированной средней интенсивности ZsYellow в AmCyan-позитивных клетках на компенсированное автофлуоресцентное среднее значение AmCyan в этих клетках. Нормализованные значения, полученные в результате одного эксперимента, были потом поделены на отношение ZsYellow/AmCyan в негативных контрольных образцах. В экспериментах оценивания ДНФ, отношения были приведены к минимальному значению. Такой же анализатор использовался в экспериментах оценивания КНФ с помощью репрессора LacI. Уровень AmCyan измерялся 405 нм лазером и 525/50 эмиссионным фильтром и dsRed-мономером с 488 нм лазером и 575/26 эмиссионным фильтром. Для каждого образца нормализованный уровень dsRed вычислялся делением компенсированного среднего значения интенсивности dsRed в AmCyan позитивных клетках на компенсированные средние значения AmCyan в этих клетках. Для анализа оценивания С1 использовался сортировщик клеток MoFlo (Darko), а также репрессор LacI-KRAB. Уровень ZsYellow измерялся 488 нм лазером и 530/40 эмиссионным фильтром, а также мономером dsRed с 568 нм лазером и 630/30
эмиссионным фильтром. Информация, полученная от оценивания С1, обрабатывалась описанным выше способом. Все полученные результаты – среднее значение 2 независимых экспериментов; по меньшей мере, 30 000 наблюдений проводилось для каждого образца. Приложение ВA universal RNAi-based logic evaluator that operates in mammalian cells