XB-IMG-174700
Xenbase Image ID: 174700
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2.3. RT-qPCRTotal RNA extraction from slc7a5 MO- or control MO-injected em-bryos (both sides) at st.15 was performed with ISOGEN (NIPPON GENE,Tokyo, Japan) and reverse transcription was carried out withSuperScript III (Thermo Fisher Scientific, Yokohama, Japan) using0.1μg of total RNA and oligo dT primer. Sequences of primer sets wereas follows:chrd.1forward, 5′-TGG ATG GTC TGC ACA ATG TT-3′;chrd.1reverse, 5′-GAA TCC AAG GTG GCA AGG TA-3′;shhforward, 5′-GCA CCA GGT CGT TCA AGT CT-3′;shhreverse, 5′-GGC AGT TAG AGGCGC ATA AG-3′;ptch2forward, 5′-GCC AGC AAG GAT CCA AAT TA-3′;ptch2reverse, 5′-CTT CTG CTC TTG GCA GGT TC-3′;foxa2forward, 5′-TCA TGG ACC TGT TCC CTT TC-3′;foxa2reverse, 5′-GAA TCA GGGTGT AGG GTC CA-3′;gli1forward, 5′-ACC AAC AGT GGG GAT GAT GT-3′;gli1reverse, 5′-TCT TGA GAG CTT GGG CTC AT-3′;gli2forward, 5′-CTC CAT GCT CAC AAC ATT GG-3′;gli2reverse, 5′-CCC AGG ACC CTAACG TCT TT-3′;Histone H4forward, 5′-TAT CAC TAA ACC CGC CATCC-3′;Histone H4reverse, 5′-TCT TCC TCT TGG CGT GTT CT-3′.Reactionwas performed with SYBR Select Master Mix and 7300 RealTime PCR System (Applied Biosystems) according to the manufacturer'sinstruction. The data was analyzed using 7300 System SequenceDetection Software Version 1.4.2.4. Whole-mount in situ hybridizationWhole-mountin situhybridization was performed as describedpreviously (Katada and Sakurai, 2016). Briefly, the method of Shainand Zuber was employed (Shain and Zuber, 1996) and embryos aftersignal detection were bleached with a solution containing hydrogenperoxide (Koga et al., 2007; Mayor et al., 1995).slc7a5.S,slc3a2.L,shh,ptch2,foxa2(also known asHNF-3β),gli1andgli2were obtained byPCR cloning using following primer sets after reverse transcription oftotal RNAs fromXenopusembryos and these amplified PCR fragmentswere subcloned intopBIISK+vector.slc7a5.Sforward, 5′-GGG GGA TCC ATG GCA GCG GGC AGC GTGAA-3′;slc7a5.Sreverse, 5′-GGG CTC GAG TTA AGA CTC CTG AGG GACAG-3′;slc3a2.Lforward, 5′-GGG GAA TTC CAA GAC ACC AGG ACTCCG AC-3′;slc3a2.Lreverse, 5′-GGG CTC GAG TTA TCC ACT ATA AGGGTA TT-3′;shhforward, 5′-GGG GGA TCC ATG CTG GTT GCG AAC TCGAA-3′;shhreverse, 5′-GGG GAA TTC TCA ACT GGA TTT CGT TGC CA-3′;ptch2forward, 5′-GGG CTC GAG CCG AAA CCA ACG TTC AGT AC-3′;ptch2reverse 5′-GGGTCT AGA CTA GTG TTG AAC AGA CAA GT-3′;foxa2forward, 5′-GGG GAA TTC ATG CTT GGG GCT GTG AAA AT-3′;foxa2reverse, 5′-GGG CTC GAG CAT GGG ACA GGA CAG AAT GG-3′;gli1forward, 5′-GGG GAA TTC TGT GAT TAC CAA GGG CAA CA-3′;gli1reverse, 5′-GGG CTC GAG ACA TTG TGT TTA GGT ACT TA-3′;gli2forward, 5′-GGG GGA TCC GTC TGT ACA GAG GAA TAT CA-3′;gli2reverse, 5′-GGG GAA TTC TTA GGA CAT CAG ATT GAG AA-3′.sox2,xk81a1,tubb2b,chrd.1andpax6are kind gifts from Dr. TsutomuKinoshita (Rikkyo University, Tokyo). The antisense probes forslc7a5.S(BamHI/T7),slc3a2.L(EcoRI/T7)sox2(EcoRI/T7),xk81a1(EcoRI/SP6),tubb2b(BamHI/T3),chrd.1(EcoRI/T7),shh(BamHI/T7),ptch2(XhoI/T3),foxa2(EcoRI/T7),gli1(EcoRI/T7),gli2(BamHI/T7) andpax6(EcoRI/T7) were prepared with restriction enzyme and RNA poly-merase shown in the parenthesis in presence of digoxygenin-UTP(RöcheDiagnostics).2.5. HistologyEmbryos after whole-mountin situhybridization were dehydratedand embedded in paraffin, Parabett (Muto Pure Chemicals, Tokyo,Japan). Sections were cut at 10μm. Slides were deparaffinized andmounted with 50% glycerol in PBS. For hematoxylin and eosin staining,sections were cut at 10μm and slides were processed according to themanufacturer's instruction (Wako Pure Chemical Industries, Osaka,Japan).Fig. 4.slc7a5 MO specifically inhibits translation ofslc7a5.Sandslc7a5.LmRNA.(A) The sequence of slc7a5 MO used in this study. slc7a5MO completely matches toslc7a5.S, but has three mis-matches toslc7a5.L. Two rescue forms,slc7a5.S rescueandslc7a5.L rescue, have 10 or 11 mismatches to slc7a5MO with silent mutations. ATG in red represents a startcodon and double dots represent the mismatch of thebase. (B–J) Validation assay of slc7a5 MO with GFP fu-sion constructs,slc7a5.S-GFPandslc7a5.L-GFP.GFPconstruct (0.5 ng) with or without MO (20 ng) was in-jected into dorsoanimal region of 4-cell stage embryos.GFPfluorescence was observed in the embryos injectedwithGFPonly orGFPand control MO, but slc7a5 MOabolished thefluorescence in bothslc7a5.S-GFPandslc7a5.L-GFPco-injection. (For interpretation of the re-ferences to color in thisfigure legend, the reader is re-ferred to the web version of this article.) Image published in: Katada T and Sakurai H (2019) Copyright © 2019. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |