Visualization of Mouse Neuronal Ganglia Infected by Herpes Simplex Virus 1 (HSV-1) Using Multimodal Non-Linear Optical Microscopy
Figure 1
Production of a recombinant strain of HSV-1 expressing a red fluorescent protein.
The region between the viral genes Us7 and Us8 was chosen as the site of insertion of an expression cassette for the red fluorescent protein mCherry. (A) The arrows indicate the open reading frames (ORF) of viral genes present in the locus. The area of interest modified by PCR involves the end of the transcribed region of the viral genes Us5, Us6, and Us7, as well as the beginning of the transcribed region of the viral gene Us8, both indicated by blue boxes. The red box indicates the location of the Poly A signal shared by Us5, Us6, and Us7. The green box indicates the location of the Us8 start codon. The line between the Us5-7 and the Us8 boxes indicates the intergenic site flanked by both transcribed regions. (B) The intergenic region was duplicated in tandem following amplification by two PCR reactions. The grey boxes represent each of the fragments generated by PCR. The restriction sites embedded within the primers used for the PCR are indicated at the ends of each fragments. (C) The transfer vector was produced following the insertion of a mCherry expression cassette between the duplicated intergenic region of Us7 and Us8. The thin grey rectangles represent the PCR fragments. The thin green rectangle represents the mCherry expression cassette. The blue boxes represent the viral ORFs of genes Us5, Us6, Us7, and Us8. The green box represents the CMV promoter. The red box represents the mCherry ORF. Diagrams are not to scale.