Establishment of HPRT and DHFR Gene Mutation Assays as biomarkers In Sheep lung fibroblasts (SLF)

Abstract

The aim of this research is to establish a gene mutation assay for examining the integrity of animal cell genome for nuclear transfer technique. Lung fibroblasts which were expanded from 4 months old female lamb were selected as target cells. These cells were coded (SLF) as Sheep lung cells. Growth characterization, doubling time, chromosomal number and structural integrity were checked after their growth in RPMI-1640 medium. For HPRT-gene mutation assay, the cells were plated at density of 1×103cells/plate and grown in medium containing toxic concentrations of 6-thioguanine; while for DHFR -gene mutation assay, toxic concentration of methotrexate was used as a selective agent. Those cells were grown for 15 days; mutant colonies either 6TGr or MTXr were reinoculated in a selective medium for further 8 weeks for checking the stability of phenotypic expression of mutant cells. The results revealed that SLF cells has spontaneous frequencies of HPRT, and DHFR gene mutations equal to 16 and 22×10-3 event/ generation/ cell, respectivel.These levels are normal comparing with other animal cell types, and these assays could be applied on other somatic cells.