Myogenic Stem Cell-Dependent Regeneration in Fish Figure 1. Schematic illustration of S. macrurus and the amputation protocol used in the present study. Tails were amputated at the end of the ventral fin (scissors). At the site of amputation, vertebrae, central artery, motor neurons, electrocytes and muscle fibers are present. The spinal cord contains two distinct populations of motor neurons: electromotoneurons that innervate electrocytes and somatomotoneurons that innervate muscle fibers. Muscle fibers are located peripherally and surround a central core of electrocytes. Electrocytes are up to 3 mm in length with a cross sectional area up to 30 times that of the adjacent muscle fibers. As shown in the cross sectional view at the bottom, muscle fibers and electrocytes are separated into distinct compartments. In this study, we analyzed the most-distal segment (50 mm) of the tail stump after tail amputation to investigate effects of amputation on tissues immediately adjacent to the injury site. doi:10.1371/journal.pone.0036819.g001 previous studies with S. macrurus myogenic tissues [31–33]. To isolate total RNA from distal-most (50 mm) tail stumps and regeneration blastema (1- and 2-week), we used the RNeasy kit (Qiagen, Valencia, CA). To remove residual DNA, total RNA isolated from each tissue sample was treated with DNase I, Amplification Grade (Ambion, Austin, TX) and analyzed by spectrophotometry (OD260/OD280). On average, total RNA isolations yielded 1–3% of starting material from each of the different tissues. All RNA samples were stored at 280uC. Cloning and Isolation of S. macrurus Pax7 Heterologous degenerate oligonucleotide primers were designed for Pax7 based on respective GenBank vertebrate sequences using Dialign and CODEHOP. Pax7 primers corresponding to the PAPGQNYPRT (sense primer) and GKKDDDDDC (antisense primer) domains were designed using alignments from protein sequences of Danio rerio (GenBank: NP_571400), Salmo salar (GenBank: CAF02090.1), Tetraodon nigroviridis (GenBank: CAG07040.1), and two sequences from Salvelinus alpinus (GenBank: CAG25716.1; CAG25714.1). To clone partial Pax7 cDNAs from S. macrurus, we carried out reverse transcription (RT) and PCR separately. The RT reaction from 250–300 ng total RNA was performed for 50 min at 42uC using the SuperScriptTM First- Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA). PCR amplification (1 mg of 1-week regeneration blastema cDNA) was carried out using PCR with annealing temperature 5uC below the calculated Tm for 35–40 cycles with PlatinumH Taq DNA Polymerase (Invitrogen). RT-PCR products for Pax7 were PCR purified by gel extraction (Qiagen, Valencia, CA), subcloned into the Topo TA2.1 Plasmid (Invitrogen) or pGEMTeasy (Promega, Madison, WI) and trans- formed into Mach1 cells (Invitrogen) or JM109 cells (Promega). Plasmids from 10–20 cDNA clones of each transcript were isolated using the QIAprepH Spin Miniprep Kit (Qiagen), sequenced in both directions using a Li-Cor 4200 L Global IR2 DNA Sequencer or an Applied Biosystems automated DNA sequencer (Model 3100), and analyzed using the Vector NTI Suite 8.0 software (InforMax, Inc., Bethesda, MD). Upon verification of the cloned sequences obtained from 1-wk cDNA regeneration blastema, nondegenerate primers for the Pax7 transcript specific to S. macrurus were generated from the on-line Primer3 software program (http://frodo.wi.mit.edu/ cgi-bin/primer3/primer3_www.cgi). Molecular Cloning of Full-length Pax7 from S. macrurus RACE first-strand synthesis from total RNA (1 mg) of 1-week regeneration blastema was performed using the BD SMARTTM RACE cDNA Amplification Kit (BD Biosciences). Pax7-specific primers used for 59-RACE were 59-GCACACTCCGTCCTTCAG- CAGCTTG-39 and 59 - GCTGGAATGGCTACTTTACCG 39, and for 39-RACE were 59-GAGACGGGTTCGATTCGTCCTG- GAG–39 and 59– TAGCCAGGTAGTCCACAGCAC –39. Purified products from Pax7 RACE experiments were subcloned into the pCRH 2.1-TOPOH vector (Invitrogen), transformed into One ShotH MACH1TM T1 Phage-Resistant Chemically Competent E. coli cells (Invitrogen), isolated with the QIAprepH Spin Miniprep Kit (Qiagen), and sequenced in both directions using either a Li-Cor-4200 L Global IR2 DNA or an Applied Biosystems automated DNA sequencer (Model 3100). Chromatogram traces were analyzed using the Vector NTI Suite 8.0 software (InforMax Inc.). Searches were performed using the Basic Local Alignment Search Tool (BLAST) network service from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). Multiple se- quence alignments were generated with ClustalW from the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw). The novel sequence described here and a transcript variant were submitted to NCBI and assigned accession numbers EU624121.2 and EU624121.1 for S. macrurus Pax7. Western Blotting Fresh-frozen tissue samples were pulverized with a mortar and pestle in liquid nitrogen and total protein lysates were prepared using lysis buffer (1% Igepal CA-630, 0.5% sodium deoxycholate, PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e36819