|
| Status |
Public on Nov 20, 2018 |
| Title |
Fitness_in vivo_+Dox_rep2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Inoculum made of Candida albicans pooled strains at a concentration of 5x10E7 cell/200 µl
|
| Organism |
Candida albicans |
| Characteristics |
strain/background: CEC2908
experiment type: in vivo
|
| Treatment protocol |
In vitro: Absence or presence of 40 μg/ml doxycycline in culture medium (see growth protocol).
In vivo: Nine- to twelve-week-old female BALB/c mice (Charles River, L'Arbresle, France) were given drinking water containing 5% sucrose, 0.1 mg/ml gentamycin and 2 mg/mL streptomycin and was supplemented or not with 2 mg/ml doxycycline, during the course of the whole experiment (14 days). Mice were housed by groups of 3 individuals per cage and were inoculated by gavage with 5x10^7 C. albicans cells (in 200 µl 1X phosphate-buffered saline (PBS) solution) from 579-strain pools at day 4 post-antibiotic treatment. The remaining inoculum was directly used for genomic DNA extraction using the MasterPure Yeast DNA Purification Kit (Epicentre). Stool samples were collected at days 4 and 10 post-gavage, weighed and either i) homogenized then serially diluted in 1 ml 1X PBS (2 stool pellets) for CFU counting on YPD plates supplemented with 1 g/l chloramphenicol and 50 mg/l gentamycin or ii) directly processed (up to 500 mg of stool pellets) for genomic DNA extraction using FastDNA SPIN Kit for Feces according the manufacturer's instructions (MP Biomedicals, cat.# 6570).
|
| Growth protocol |
In vitro conditions: An aliquot (1.6 µl) from the frozen 50 OD600-unit strain-pool was used to inoculate 2 ml of YPD medium (starting OD600 = 0.0625) and grown at 30ºC with agitation (200 rpm) for 20 generations, in the absence or presence of 40 μg/ml doxycycline.
In vivo conditions: See treatment protocol.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
In vitro fitness experiments: Genomic DNA was extracted using the MasterPure Yeast DNA Purification Kit (Epicentre) as per the manufacturer's instructions.
In vivo fitness experiments: Total genomic DNA from stools was extracted using FastDNA SPIN Kit for Feces according to the manufacturer’s instructions (MP Biomedicals, cat.# 6570). Barcodes were amplified from genomic DNA by PCR using common primers flanking each barcode sequence.
|
| Label |
Cy3
|
| Label protocol |
A modified base [5-(3-aminoallyl)-dUTP] was included during PCR at a ratio of two 5-(3-aminoallyl)-dUTP molecules to three dTTP molecules. The PCR products were purified, and the DNA was labeled with either Cy3 (for the in vitro experiments, untreated control; for the in vivo experiments, inoculum) or Cy5 (for the in vitro experiment, Dox-treated samples; for the in vivo experiments, stools from dox-treated or dox-untreated mice) monoreactive NHS esters.
|
| |
|
| Channel 2 |
| Source name |
Stools produced from dox-treated mice at day 10 post-gavage with an inoculum of Candida albicans pooled strains at a concentration of 5x10E7 cells/200 µl
|
| Organism |
Candida albicans |
| Characteristics |
strain/background: CEC2908
experiment type: in vivo
treatment: doxycycline
|
| Treatment protocol |
In vitro: Absence or presence of 40 μg/ml doxycycline in culture medium (see growth protocol).
In vivo: Nine- to twelve-week-old female BALB/c mice (Charles River, L'Arbresle, France) were given drinking water containing 5% sucrose, 0.1 mg/ml gentamycin and 2 mg/mL streptomycin and was supplemented or not with 2 mg/ml doxycycline, during the course of the whole experiment (14 days). Mice were housed by groups of 3 individuals per cage and were inoculated by gavage with 5x10^7 C. albicans cells (in 200 µl 1X phosphate-buffered saline (PBS) solution) from 579-strain pools at day 4 post-antibiotic treatment. The remaining inoculum was directly used for genomic DNA extraction using the MasterPure Yeast DNA Purification Kit (Epicentre). Stool samples were collected at days 4 and 10 post-gavage, weighed and either i) homogenized then serially diluted in 1 ml 1X PBS (2 stool pellets) for CFU counting on YPD plates supplemented with 1 g/l chloramphenicol and 50 mg/l gentamycin or ii) directly processed (up to 500 mg of stool pellets) for genomic DNA extraction using FastDNA SPIN Kit for Feces according the manufacturer's instructions (MP Biomedicals, cat.# 6570).
|
| Growth protocol |
In vitro conditions: An aliquot (1.6 µl) from the frozen 50 OD600-unit strain-pool was used to inoculate 2 ml of YPD medium (starting OD600 = 0.0625) and grown at 30ºC with agitation (200 rpm) for 20 generations, in the absence or presence of 40 μg/ml doxycycline.
In vivo conditions: See treatment protocol.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
In vitro fitness experiments: Genomic DNA was extracted using the MasterPure Yeast DNA Purification Kit (Epicentre) as per the manufacturer's instructions.
In vivo fitness experiments: Total genomic DNA from stools was extracted using FastDNA SPIN Kit for Feces according to the manufacturer’s instructions (MP Biomedicals, cat.# 6570). Barcodes were amplified from genomic DNA by PCR using common primers flanking each barcode sequence.
|
| Label |
Cy5
|
| Label protocol |
A modified base [5-(3-aminoallyl)-dUTP] was included during PCR at a ratio of two 5-(3-aminoallyl)-dUTP molecules to three dTTP molecules. The PCR products were purified, and the DNA was labeled with either Cy3 (for the in vitro experiments, untreated control; for the in vivo experiments, inoculum) or Cy5 (for the in vitro experiment, Dox-treated samples; for the in vivo experiments, stools from dox-treated or dox-untreated mice) monoreactive NHS esters.
|
| |
|
| |
| Hybridization protocol |
The Cy3- and Cy5-labeled DNAs were mixed in 50 µl of DIG Easy Hyb solution (Roche) and hybridized overnight at 24°C to a barcode microarray.
|
| Scan protocol |
Images of Cy5 and Cy3 fluorescence intensities were generated by scanning the barcode arrays using an Axon Autoloader 4200AL scanner (Molecular Devices, Downington, PA). Images were subsequently analyzed with the GenePix Pro 6.1.0.2 software (Molecular Devices, Downington, PA).
|
| Description |
Sample 5
Background strain is BWP17 (ura3-/-, arg4-/-, his1-/-) complemented for arg- and his- auxotrophies (his1-delta::hisG/HIS1, arg4-delta::hisG/ARG4) and carrying the pNIMX plasmid (ADH1/adh1::PTDH3-carTA::SAT1). The resulting strain is CEC2908, isogenic to CEC2907 described in Chauvel et al. 2012 PLOS ONE 7(9): e45912.
|
| Data processing |
GenePix Results (GPR) files were imported into Arraypipe 2.0 (http://koch.pathogenomics.ca/cgi-bin/pub/arraypipe.pl). Following background subtraction, spot filtering and bad-spot flagging, global signal intensities were normalized using Loess normalization. Replicate slides were combined and Z-scores and P-values (standard Student’s t-test) were calculated.
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| |
|
| Submission date |
Mar 24, 2015 |
| Last update date |
Nov 20, 2018 |
| Contact name |
Sadri ZNAIDI |
| E-mail(s) |
sadri.znaidi@gmail.com
|
| Organization name |
Institut Pasteur
|
| Department |
Mycology Department
|
| Lab |
Fungal Biology and Pathogenicity
|
| Street address |
25 rue du Docteur Roux
|
| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
| |
|
| Platform ID |
GPL17420 |
| Series (2) |
| GSE67215 |
Fitness assay of Candida albicans strains grown in vitro or in vivo in a mouse model of gastrointestinal colonization |
| GSE67235 |
Systematic gene overexpression in Candida albicans identifies a regulator of early adaptation to the mammalian gut |
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