|
| Status |
Public on Aug 27, 2018 |
| Title |
miR202 knock-out Female 17 |
| Sample type |
RNA |
| |
|
| Source name |
miR202 knock-out Female
|
| Organism |
Oryzias latipes |
| Characteristics |
treatment: KO miR202
genotype: miR202 knock-out
tissue: ovary
|
| Treatment protocol |
For the CRISPR/Cas9 strategy, the target genomic sequence was identified with the help of the ZiFiT online tool (http://zifit.partners.org/ZiFiT/) and using the medaka genome reference available on the Ensembl genome database (Ensembl gene: ENSORLG00000021212). A short sequence in the mature miR-202-3p was selected as followed: GG-(N)18-NN. Two inverse-complementary primers (Forward 5’-TAGGCATAAGGCATGGGAAAAT-3’ and Reverse 5’-AAACATTTTCCCATGCCTTATG-3’) were annealed and cloned into the pDR274 vector (Addgene plasmid 42250). The modified pDR274 vector was digested with DraI and the miR-202 specific guide RNA (mir202-sgRNA) was transcribed using the T7 RNA polymerase (P207, Promega). For the Cas9-RNA in vitro synthesis, the pCS2-nCas9n vector (Addgene plasmid 47929) was linearized with NotI and capped RNA encoding the Cas9 was transcribed with the mMessage mMachine SP6 Kit (AM1340, Life Technologies) following manufacturer's instructions. Cas9-RNA (100 ng/μL) and mir202-sgRNA (10 ng/μL) were co-injected into one-cell stage embryos. Injected embryos were raised to sexual maturity and 10 fishes were genotyped to identified founder fishes (F0). Fishes harboring the same INDEL mutation (-7+3) were selected and outcrossed with wild-type fishes to obtain F1 heterozygous. Such outcrosses were performed at each generation in order to maintain the line.
|
| Growth protocol |
Adult medaka (Oryzias latipes) from the CAB strain was used. Wild-types, miR-202 mutants and transgenics Tg(sox9b::EGFP) were raised at 26°C under a growing photoperiod regime before 3 months old (12h light/ 12h dark) or under a reproduction photoperiod regime after 3 months old (14 h light/10 h dark)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen tissues were lysed with Precellys Evolution Homogenizer (Ozyme, bertin technologies) in TRI Reagent® (TR118, Euromedex) and total RNA was extracted using the “nucleospin RNA” kit (740955, Macherey Nagel).
|
| Label |
Cy3
|
| Label protocol |
Labeling and hybridization steps were performed following the “One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling)”Agilent protocol. Briefly, for each sample, 150 ng of total RNA was amplified and labeled using Cy3-CTP. Yield (>0.825µg cRNA) and specific activity (> 6pmol of Cy3 per µg of cRNA) of Cy3-cRNA produced were checked with the Nanodrop.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-cRNA was fragmented and hybridized on a sub-array. Hybridization was carried out for 17 hours at 65°C in a rotating hybridization oven prior to washing.
|
| Scan protocol |
Microarrays were scanned with an Agilent Scanner (Agilent DNA Microarray Scanner, Agilent Technologies, Massy, France) using the standard parameters for a gene expression 8x60K oligoarray (3µm and 20bits)
|
| Data processing |
Data were then obtained with the Agilent Feature Extraction software (10.7.3.1) according to the appropriate GE protocol (GE1_107_Sep09). Normalization and statistical analysis were performed using GeneSpring software (Agilent).
|
| |
|
| Submission date |
Mar 05, 2018 |
| Last update date |
Aug 27, 2018 |
| Contact name |
Aurelie LE CAM |
| E-mail(s) |
aurelie.le-cam@inrae.fr
|
| Organization name |
INRA
|
| Department |
PHASE
|
| Lab |
LPGP
|
| Street address |
Campus de Beaulieu
|
| City |
Rennes |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL24100 |
| Series (1) |
| GSE111388 |
The microARN-202 (miR-202) controls female fecundity by regulating oogenesis in medaka |
|
