Chicken embryo fibroblasts (CEF) and Marek’s disease virus infection
Twenty-one chicken embryos (9-days old) from SPF eggs for preparation of chicken embryo fibroblast (CEF) were from SPAFAS Co. (Jinan, China; a joint venture with Charles River Laboratory, Wilmington, MA, USA). Each embryos was trypsinized according to conventional procedures, and the cells were suspended in Medium 199 supplemented with 5% calf serum, glutamine (2 mM), sodium pyruvate (1 mM), Penicillin (1000 u/mL) and Streptomycin (1000 μg/ml) in a 75 cm2 flask (Corning Glass Works, Coming, NY). The fibroblasts from each chicken embryo were divided into two for in vitro culture. One is assigned for control and another for infection with the virulent RB-1B strain of MDV. The RB-1B strain of MDV was provided by Dr. Aijian Qin (College of Veterinary Medicine, Yangzhou University, China).
At day 5 post infection of MDV, 95% of CEF showed cytopathic effect (CPE). The control fibroblasts and MDV-infected CEFs were used for DNA exraction. The cells were scraped from the flasks and pelleted by centrifugation. The pellet was washed once with 0.2 mol/L pH 7.2 phosphate buffered saline and pelleted again. The pellet was lysed in digestion buffer (10 mM Tris–Cl pH 8.0, 0.1 M EDTA pH 8.0, 0.5% SDS, 20 μg/mL pancreatic RNase) and treated with proteinase K. After extraction with phenol, DNA was precipitated with ethanol, dissolved in 1X TE (10 mM Tris–Cl pH 7.5, 1 mM EDTA). The concentration of DNA was determined by OD260 using the BioPhotometer plus (Eppendorf, Germany). The quality of DNA was checked by agarose gel electrophoresis.
Primers, PCR and gel electrophoresis
One-hundred-eighteen microsatellite markers in chicken linkage map
 were chosen to determine MSI in present study (Table
1). Each microsatellite repeat was amplified by polymerase chain reaction (PCR) and subjected to gel electrophoresis aimed at comparing the pattern of MDV-infected DNA and control DNA from the same chicken embryo fibroblasts. All primers were provided by Dr. Takahashi, National Institute of Agro biological Sciences (NIAS), Tsukuba, Japan.
PCR was performed in a 12.5μL reaction mixture containing 0.5 μl template DNA (100 ng), 0.5 μl of Taq DNA polymerase (2.5 U/μl), 1.25 μl of 10 × PCR buffer, 0.5 μl of each primer (5 μmol/L), 1 μl of dNTPs (2.5 mmol/L), 8.25 μl distilled H2O. PCR was performed as following steps: a hot start, 94°C for 15 s, then 94°C for 15 s, 55°C for 30 s, and 68°C for 60 s, all repeated for 10 cycles, followed by 10 under the same condition except for the annealing temperature that was changed to 50°C and 45°C, finally an elongation time of 9 min at 68°C. PCR amplification was performed using the PTC-200 (Bio-Rad, USA). PCR products were separated by electrophoresis in 12% polyacrylamide gel containing 6 M urea for 2.5 h at 70 W. Gels were fixed in 10% acetic acid, and stained with AgNO3.
Each gel was compared with matched normal and MDV-infected CEF specimens. To accurately size the PCR products, a DNA ladder was used on both sides of the gel. Comparisons were made between the number of bands present in both MDV-infected and normal CEF samples. Band intensity was noted but was not categorized for differences between the normal and abnormal DNA. To quantify the presence or absence of MSI, each primer was analyzed for differences in the number of alleles present, be it through insertions or deletions (Figure
1). All microsatellite markers were analyzed for each MDV-infected CEF sample. If the number of allelic bands were not equal in number, band intensity had a significant difference, they were graded as MSI.