LITMUS: multipurpose cloning vectors with a novel system for bidirectional in vitro transcription.

We describe the construction and uses of a set of four multipurpose cloning vectors: LITMUS 28, 29, 38 and 39. The vectors feature the high-copy pUC origin and an M13 origin for single-stranded DNA production as well as polylinker sites for most commercially available restriction enzymes that recognize nondegenerate hexanucleotide sites and yield 4-base sticky ends upon cleavage. Sites are arranged, without overlaps, to permit linker addition to blunt-ended fragments and unidirectional nested deletions and are within the lacZ alpha gene to facilitate blue-white screening. Finally, the polylinkers are flanked by a pair of opposing modified T7 promoters to allow in vitro transcription of either strand of a cloned insert with T7 RNA polymerase. Selective unidirectional transcription from one promoter is achieved by cleaving the other at an internal restriction site (AflII or SpeI). Both modified promoters are fully active under standard RNA probe synthesis conditions. In Southern blots of Dirofilaria immitis genomic DNA, an RNA probe prepared from LITMUS performed equivalently to the same RNA probe made from a wild-type promoter vector and a DNA probe prepared by random priming.

In vitro photodynamic treatment of normal and human papilloma virus-transfected keratinocytes with photofrin II and red light.

Photodynamic therapy involves the use of light of appropriate wavelength to excite a photosensitizer to result in tissue destruction. The photosensitizer dihematoporphyrin ether and red light were used to treat normal and human papilloma virus type 18-transfected keratinocytes in vitro. Although both cell lines were sensitive to treatment, normal keratinocytes retained more dihematoporphyrin ether and were more sensitive to photodynamic therapy than were transfected cells. In vitro data fail to show the selective retention of dihematoporphyrin ether reported elsewhere in the literature in papillomas and tumors. We did not find selective uptake or retention of dihematoporphyrin ether by human papilloma virus-transfected cells, despite previously published in vivo data to the contrary. Dihematoporphyrin ether retention and thus selective photosensitivity of papillomas in vivo is not caused by individual cellular differences in the processing of dihematoporphyrin ether. In addition, because in vitro results may not parallel in vivo results, both in vivo and in vitro models must be investigated in the study of phototherapeutic agents.