Cloning of cellular miRNA sequences into NL4-3. Schematic representation (Top) of the NL4-3 genome showing the insertion of the miRNA sequences into the Nef locus. The bottom panel shows PCR analyses of the indicated molecular clones performed to verify insertion. The p398.6 clone was a shuttle vector used in transferring the cloned miRNA into the pNL4-3 full length molecular clone. PCR primers were selected that flank the insertion site in Nef. Wild-type virus (lane 1) yielded a band of 220 bp. The p398.6 shuttle vector (lane 2) yielded a band of 143 bp. Insertion of the miRNA sequences (lanes 3-10) yielded a PCR fragment size commensurate with the size of the insert. Sizes of the molecular weight ladder (M) are indicated on the left.