A HPLC-based chloramphenicol acetyltransferase assay for assessing hair growth: comparison of the sensitivity of UV and fluorescence detection.
In our attempt to measure hair growth by hair-specific markers, we used transgenic mice to express the chloramphenicol acetyltransferase gene under the control of an ultrahigh sulphur keratin gene promoter. To quantitate expression of the keratin gene, we required a chloramphenicol acetyltransferase assay which could measure enzyme activity in a single follicle and also could be used to assay a large number of samples without loss of sensitivity. We achieved this objective by utilizing a fluorescent substrate for chloramphenicol acetyltransferase. With HPLC-fluorescence detection, this substrate provides a sensitivity of less than 1 x 10(-13) mol, which is 1000 times greater than that achievable with HPLC-UV detection in cultured follicles. Further, the assay was automated to facilitate the analysis of more than 100 samples/day. It should be possible to apply this fluorescent assay to a number of cell or tissue studies.
Waldon
D J
DJ
Dermatology Department, Upjohn Company, Kalamazoo, MI.
Kubicek
M F
MF
Johnson
G A
GA
Buhl
A E
AE
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D046508:Culture Techniques; D015870:Gene Expression; D006197:Hair / Q000254:growth & development*; D007633:Keratins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D007633:Keratins; D015500:Chloramphenicol O-Acetyltransferase