FIG. 1.
Structure of the HSV-1 LAT transgene and analysis of its copy number in transgenic mice. (A) Structure of the HSV-1 LAT transgene. (I) Schematic representation of HSV-1 genome and LAT transcripts. The unique long (UL) and short (US) regions are represented as lines, and the internal and terminal long and short repeats (IRL, TRL, IRS, and TRS) are represented as open boxes. The locations of the LAT gene and its 8.3- and 2.0-kb LATs are indicated. (II) DNA fragment inserted into mice. The 2.0-kb LAT gene, the locations of the CMV promoter and simian virus 40 (SV40) poly(A) site, the relevant restriction sites, and the location of the BstEII-cleaved LAT gene probe are indicated. (III) Locations of the primers used for RT-PCR and for quantitative RT-PCR. The arrows indicate the two colinear 2.0- and 1.5-kb LATs and their orientations. (B) LAT copy number analysis. DNAs (5, 10, and 20 μg), extracted from tail biopsy specimens from transgenic mice, were slot blotted to two different filters: one was hybridized with the LAT DNA probe (BstEII cleaved; 977 bp [panel A, II]), while the other filter was hybridized with a mouse α-actin cDNA fragment (PstI cleaved; 1,100 bp) (27) as a diploid gene control (LAT and α-actin, respectively). As a negative control, the same amounts of DNA derived from nontransgenic mice were blotted to the filters. The specific activities of the two probes were similar (3 × 109 ± 0.3 × 109 cpm/μg of DNA). Computerized image analysis and quantification of radioactive signals were performed with a Bio-Imaging analyzer (45). The LAT transgene copy numbers were estimated relative to the α-actin radioactive signal and are indicated above the columns.