Design and Development of a Cell Marking System in Transgenic Mice

The aim of this project was to develop a marker system to analyse cell lineage in mouse skin. This should provide useful information on the usual size and organisation of clones in the epidermis and give some clues as to how cell replacement is normally controlled in the epidermis and to the possible location of the stem and progenitor cells. As there is no marker gene currently available in the murine epidermis a marker gene had to be designed and introduced. This was accomplished by generating transgenic mice with a novel marker gene inserted into their DNA. An Escherichia coli lac Z gene was used as a cell marker and activated in single cells following treating the mice with a chemical mutagen. The marker could be activated by a point mutation in the sequence of an oligonucleotide inserted at the 5' end of the lac Z gene, replacing the ATG initiation codon of the gene. Two different marking strategies were devised. One specific for the chemical mutagen N-nitro-N'-methyl-N-nitosoguanidine (MNNG) and the other for the chemical mutagen acetoxyaminofluorene (AAF). Oligonucleotides were designed, synthesised and cloned in front of the lac Z gene to generate oligollac Z fusion marker genes. Test constructs without initiation codons and control constructs with initiation codons were designed and cloned at the same time for both the MNNG and AAF strategies. The fusion genes were then tested in vitro by transfection in to murine epidermal cell lines. No expression was detected in the inactive marker gene designed to be activated by MNNG, whereas high expression was obtained from two control constructs which both contained and in-frame ATG initiation codon in the sequence of the oligonucleotide cloned into the lac Z gene. The MNNG-responsive marker gene was shown to be activated in stably transfected murine epidermal cell lines in response to treatment with MNNG at a frequency of approximately 4.3 x 10-3 per clonogenic cell. Subsequently the work with the AAF strategy was discontinued as there was insufficient time to pursue both strategies and positive results had been obtained with the MNNG construct. Twenty-two transgenic founders were generated with three marker gene constructs (Act-LacZA, K5-LacZD and K5-LacZE by pronuclear microinjection of fertilised embryos. Lines of transgenic mice were bred from the founders and the expression of the marker genes were analysed. Expression from a control lac Z fusion gene driven by a BKIII (keratin 5) promoter was detected by staining skin tissue with the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal). The expression of the beta-galactosidase protein was observed in the epidermal basal layer and the hair follicles of transgenic mice. The expression was highest in newborn mice and gradually diminished as the mice reached adulthood. It was subsequently shown that targeting the expression of an activated H-ras oncogene to the epidermis with the same promoter results in a high frequency of malignant tumours. The implications of these results to the biology of the skin are discussed. Expression of the MNNG-activated test marker gene under the control of a beta-actin promoter was also detected. Activation of the marker gene was detected in serial sections of murine brain tissue from transgenic mice treated topically with MNNG at birth. No activation was detected in other tissues probably due to the low level of expression of the marker gene. The explanation of these results and the implications for developing a marker system to work efficiently in the epidermis are discussed