Exogenous gene expression in mice treated with primary B cells infected with SNV-MPL vector virus expressing PLP.

(A) A diagram of the MPL cassette is depicted. Recognition sites for the restriction enzymes StuI and BglII are indicated as well as the positions of the primers 5′-M6PR and 3′-M6PR used for RT-PCR. (B) RT-PCR amplification was performed on total RNA extracted from the spleens of protected (lanes 2-11) and unprotected (lanes 12-14) mice reconstituted with SNV-MPL–infected B cells. PA and TD indicate paralysis and tail drop, respectively. Lane 1 represents SNV-MPL infected, neo-selected WEHI 231 cells as positive control and lane 15 represents spleen from an untreated normal control mouse. The amplification of the housekeeping gene GAPDH was performed in each sample to ensure that equal levels of cDNA were used. (C) To confirm that the amplified sequence corresponds to the exogenous sequence, some of the amplified products were purified and analyzed by restriction enzyme digestion. Lanes 1 and 3 represent the DNA from a protected mouse and lanes 2 and 4 represent DNA from the SNV-MPL–infected WEHI 231 cell line. Lanes 1 and 2 were digested withBglII and lanes 3 and 4 were digested with StuI. Both enzymes cut once within the amplified sequence and yielded the expected size bands.