Mice were housed in open-topped cages with autoclaved pine shavings while bedding and were fed autoclaved proprietary mouse chow (Specialty Feeds, Glen Forrest, Australia) prepared to withstand autoclaving and acidified (pH 2

Mice were housed in open-topped cages with autoclaved pine shavings while bedding and were fed autoclaved proprietary mouse chow (Specialty Feeds, Glen Forrest, Australia) prepared to withstand autoclaving and acidified (pH 2.6) D-Luciferin potassium salt water. 0.15 mM dNTPs, 0.15 M MPV1_3505F, 0.15 M MPV1_4158R, and 0.046 U/L Platinum polymerase (Invitrogen). The PCR cycling consisted of 1 cycle of 94 C for 3 min followed by 30 cycles of 94 C for 30 s, 54 C for 30 s, and 72 C for 30 s, with 1 final cycle of 72 C for 7 min. The PCR reaction products were separated by electrophoresis on a 1% agarose Rabbit Polyclonal to TBC1D3 gel, stained with ethidium bromide, and visualized by using a UV transilluminator. Visual images were produced by using Molecular Analyst software version 1.4 (Bio-Rad). DNA sequencing and analysis. DNA extracted from spleen tissue of an Hsd:NIH mouse identified as PCR-positive for MPV1 was used as a template for sequencing of the virus. This isolate provisionally was designated MPV1f. The entire genome but excluding the terminal palindromic regions was amplified for cloning by using 5 sets of primers (Table 1) to produce 5 overlapping amplicons. The reaction conditions were similar to those used for detection of viral infection. Each product was separated by using agarose gel electrophoresis and purified from the gel by using the Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI). These products were cloned into the pGEM-T Easy vector (Promega) according to the manufacturer’s instructions. Plasmid DNA was isolated by using the QIAprep Spin Miniprep Kit (Qiagen). The cloned products were sequenced by using either vector-specific primers or primers specific to the cloned virus DNA, with the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PerkinElmer, Waltham, MA). Each region of viral DNA was sequenced from 4 different plasmid clones, each obtained from separate PCR reactions. The resulting sequences were modified to remove sequence arising from the vector and primers D-Luciferin potassium salt and were combined by using CAP3.10 Sequence alignments and similarity plots with other MPV types were performed by D-Luciferin potassium salt using ClustalW14 and sequences obtained from GenBank. Recombinant truncated MPV1f VP1 capsid protein. Oligonucleotide primer pair MPV1_3505F and MPV1_4158R (Table 1) were based on the sequence of MPV1a (GenBank accession no., MPU_12469) and were designed to amplify a region of the VP1 gene of MPV1f that encoded the amino acids considered the primary determinants of tissue tropism of MVM, D-Luciferin potassium salt referred to as the allotropic determinants.4 Amplification of this region by PCR was performed in 20-L volumes in an automated Thermal Cycler (Bio-Rad) by using 200-L flat-top PCR tubes (Sarstedt, Nmbrecht, Germany) and commercial reagents (Invitrogen). Each reaction contained 1 L of the extracted DNA eluate as template, 0.916 U Platinum DNA polymerase, 0.2 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 10 PCR buffer, 1.5 mM MgCl2, 20 pmol/L of each oligonucleotide primer, and ultrapure water. The thermal cycling conditions were an initial cycle of 94 C for 3 min, followed by 30 cycles of 94 C for 30 s, 54 C for 30 s, and 72 C for 30 s, with a final period of 72 C for 7 min. The PCR products were electrophoretically separated in 1.2% (w/v) electrophoresis-grade agarose (Progen, Toowong, Australia) containing ethidium bromide (Invitrogen) at 80 V for 1 h by using TAE buffer (1 mM EDTA, 40 mM Tris-acetate, pH 8.0). The amplified PCR products was excised and purified by D-Luciferin potassium salt using the Wizard PCR Purification System (Promega) and ligated into the PinPoint Xa1 vector (Promega) as specified by the manufacturer. The recombinant vector was transformed into high-efficiency JM109 cells (Promega) by using a heat-shock method. The transformed cells were grown overnight on LB agar plates containing ampicillin at a final concentration of 100 g/mL. Colonies were picked by using sterile toothpicks into individual 1.5-mL.