Unveiled: Why Isotretinoin May Make Us All Happier

Additional simple sequence length polymorphism (SSLP) markers were designed based on genomic dinucleotide repeats. Recombinant mutant embryos were screened with these markers to further narrow the genomic region. Coding sequence of candidate genes within the region were cloned by RT-PCR and sequenced to uncover mutations in s231, m628, and m673 alleles. Morpholino and mRNA Microinjections, Transplantation For rescue/overexpression analysis, full-length WT or mutant (s231, m628, m673) med14 coding sequence was subcloned into pCS2+ vector for in?vitro transcription using the mMESSAGE mMACHINE Selleckchem GDC0068 kit (Applied Biosystems) and injected at 300 pg per embryo. A med14 morpholino targeting the ATG translational start site (5��-CCGAACCGATCTGAACTGGAGCCAT-3��) was purchased from Gene Tools, with 6?ng injected per embryo. For transplantation Isotretinoin experiments, donor embryos from a logm628+/?; Ola.Actb:Hsa.HRAS-EGFPvu119?+/? cross were used, with cells transplanted into multiple regions of WT host embryos at 4 hpf. Donor embryos were kept paired with corresponding host embryos to identify log mutant donors that were EGFP+��ve. RNA ISH RNA ISH using DIG-labeled antisense RNA probes was performed as previously described (Pearson et?al., 2009; Thisse and Thisse, 2008). Fluorescent ISH (FISH) in planarians using the alkaline phosphatase (AP) substrate Fast Blue was performed as previously described (Cowles et?al., 2013). Probe fragments used are described in Supplemental Information. Microarray Analysis of Gene Expression Two-color microarray experiments were performed by the UHN Microarray Facility using the Zebrafish (v.3) 44?k Gene Expression Microarray Platform (Agilent). cDNA was generated from total RNA?isolated from pools of 20 WT or m628 mutant embryos at 54 hpf, with two biological replicates used. Microarray results were analyzed using Genespring INCB28060 chemical structure v.11.0.1 (Agilent), with data normalized using Agilent��s Spatial Detrending and Lowess normalization. After normalization and averaging, data were filtered such that only probes that were between the 20th and 100th percentile of the distribution of intensities in both samples for either group were kept. Statistical significance for differential expression between sample groups was set at p?