Primary mouse anti-Myf5 (SC-302 clone C-20) and secondary goat anti-rabbit-HRP conjugated were used to detect the Myf5 specific band at 35 kD. Primary mouse anti-MyoD (BD Biosciences c# 554130), mouse anti-myogenin (BD Bioscience, East Rutherford, NJ, http://www.bd.com
c# 556358), mouse anti-M-cadherin (BD Bioscience c# 611100), mouse anti-myosin http://www.selleckchem.com/
heavy chain (MyHC [DSHB F1.652S]), and secondary anti-mouse HRP conjugated were used to detect specific bands of 45, 34, 130, 180 kD, respectively. Total RNA was extracted with Trizol (Invitrogen) and cDNA was generated using 1 ��g DNase-treated RNA with oligo-dT primers and TermoScript (Invitrogen). PCR was performed using TaqMan Real Time PCR premixtures on a 7500 RT-PCR System from Applied Biosystems. For muscle-related genes, premade probes were purchased from Applied Biosystems: Myod1 Mm00440387_m1, Myf5 Mm00435125_m1, Myog Mm00446194_m1, Mck Mm0432556_m1, Desmin Mm00802455_m1, m-Cad Mm00483183_m1, and Gapdh Mm99999915_g1. For human probes: MYOD1 Hs00159528_m1, MYF5 Hs00271574_m1, MYOG Hs01072232_m1, DESMIN Hs00157258_m1, and GAPDH Hs99999905-m1. All reactions were performed at least in triplicate and the data were analyzed by 7500 System Software (Applied Biosystems, Foster City, CA, www.appliedbiosystems.com). The difference in threshold www.selleckchem.com/products/forskolin.html
cycle number between transcripts of interest and GAPDH was used to determine the abundance of transcript relative to GAPDH (��-CT method). All experiments were done at least three times. Data shown for real time PCR are the mean �� SD. Difference between means was compared by the two-tailed Student's t test (GraphPad Prism five) and was considered significantly different at p < .05. The genomic region upstream of HPRT has been demonstrated to be a site at which transgenes express reliably [9, 16-18], presumably due to HPRT being a Saracatinib
housekeeping gene, active in all cell types and therefore embedded in constitutively open chromatin. We have targeted this site with a doxycycline-inducible cre transgene flanked by heterologous, self-incompatible loxP sites (Fig. 1A) in A17 mESCs, a derivative of E14Tg2a  in which rtTA has been inserted into the Rosa26 locus . Downstream of the floxed cre is ��neo, a G418 resistance gene lacking a start codon and promoter . These cells are referred to as A2Lox.cre. Treating cells with doxycycline causes cre to be expressed, rendering the cells competent for recombination. When transfected with p2Lox, a plasmid bearing the same heterologous loxP sites, a cassette exchange recombination replaces cre with a gene of interest from the incoming plasmid. Because the orientation of the loxP sites is inverted on the plasmid relative to the chromosome, the plasmid in its entirety (except for the short sequence between the loxP sites) is inserted.