Salmon sperm dna

A375 cells were transfected with 0.9 µg of spGFP transposon and 0.1 µg of pCDNA-mPB (piggyBac transposase) and selected with 10 µg/mL Blasticidin for 3 weeks. Clones derived from single cells were picked using cloning cylinders and expanded. Genomic DNA was isolated with Proteinase K lysis buffer and 15 µg of DNA were digested overnight with 40 units of MspI (New England Biolabs, Cat. # R0106). Digested DNA was separated on a 0.8% agarose gel, followed by in-gel depurination, denaturation, and neutralization. DNA was capillary transferred in 10xSSC onto nitrocellulose membrane and crosslinked by baking at 70°C for one hour. A Blasticidin probe was PCR amplified, labelled with α-³²P-dCTP using DECAprime II random prime labelling kit (Invitrogen, Cat. # AM1456) and purified with MicroSpin G-50 columns (Cytiva, Cat. # 27533001) using the manufacturers’ recommendations. The membrane was incubated with labelled probe and 250 µg/mL salmon sperm DNA (Roche, Cat. # 10223638103) at 65°C overnight in Perfecthyb Plus Hybridization Buffer (Millipore, Cat. # H7033-125ML), washed in 2xSSC plus 0.5% SDS, and exposed to X-ray film for 4 days.

Animals: Male Wistar rats of 8–12 weeks of age with a body weight of approximately 170–200 g were bought from Harlan–Winkelmann (Brochen, Germany). Adult male B6.129S2-Il6tm1Kopf (IL-6-KO) mice and wild-type adult male C57BL/6J mice (25–28 g) that were 8–12 weeks of age were purchased from Jackson Lab. (Bar Harbor, ME, USA). Both the rats and mice were held under standard conditions with a 12 h light–dark cycle and ad libitum access to fresh water and food. The animals were held according to the instructions of the institute, the German Animal Welfare Convention, and NIH guidelines.
Chemicals: The chemicals used in the present study were of analytical/molecular biology grade and purchased from commercial sources, as follows: the total RNA was extracted from the livers of mice after homogenization in Trizol (Invitrogen, Carlsbad, CA, USA). The primers were purchased from IBA Lifesciences (Goettingen, Germany). Quantitative real-time (qRT)-PCR primers, M-MLV reverse transcriptase, platinum Sybr green qPCR–UDG mix were obtained from Invitrogen (Darmstadt, Germany); and dNTPs, protector RNase inhibitor, Klenow enzyme, primer oligo dT15 for cDNA synthesis, and Salmon sperm DNA were obtained from Roche (Mannheim, Germany). All other reagents and chemicals were from Sigma–Aldrich or Merck (Darmstadt, Germany).

All chemicals used were of analytical grade and purchased from commercial sources as follows: real-time polymerase chain reaction (PCR) primers, moloney murine leukemia virus (M-MLV) reverse transcriptase, reverse transcription buffer, 0.1 M dithiothreitol (DTT), and Platinum SYBR green qPCR UDG mix were from Invitrogen (Carlsbad, CA, USA); dNTPs, Protector RNase inhibitor, Klenow enzyme, primer oligo (dT)15 for complementary DNA (cDNA) synthesis, and Salmon sperm DNA were from Roche (Penzberg, Germany); Hybond N nylon membranes were purchased from Amersham Pharmacia Biotech (Amersham, UK), 4,6-diamidino-2-phenylindole (DAPI) from Southern Biotech (Birmingham, AL, USA). All other reagents and chemicals were from Sigma-Aldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany).