54 THE DEVELOPMENT AND CHARACTERIZATION OF MITOCHONDRIAL DNA (MTDNA)-DEPLETED CAPRA HIRCUS FETAL FIBROBLASTS: CANDIDATE DONORS FOR SOMATIC CELL NUCLEAR TRANSFER (SCNT)

Abstract

Mammalian mtDNA is approximately 16.6 kb in size. It has 37 genes, 13 of which encode protein subunits of the oxidative phosphorylation (OXPHOS) system, the major ATP-generating pathway of the cell. Normally, mammals inherit a single mtDNA genome (homoplasmy) from their mother. Somatic cell nuclear transfer (SCNT) violates this strict maternal, homoplasmic inheritance of mtDNA as cytoplasm is transferred along with the nucleus, which often results in an oocyte harboring both donor and recipient mtDNA genomes (heteroplasmy). This been previously reported (reviewed St. John JC 2002 Theriogenology 57, 109–123). To overcome the problem of donor mtDNA transmission, we have developed and characterized mtDNA-depleted C. hircus (goat) cells for use as donors in SCNT. C. hircus primary foetal fibroblast cells were established in culture and depleted of their mtDNA by supplementing their growth medium with a low concentration, 50 ng mL⁻¹, of ethidium bromide (EthBr). Conventional PCR, using a series of primers designed specifically for goat mtDNA, was used to screen for the presence of mtDNA during the EthBr treatment. In addition, mitochondrial organization, activity and morphology in the cells were analyzed using the mitochondrial specific fluoroprobe JC1. mtDNA-encoded and mitochondrial transcription factor A (mtTFA) transcript levels were analysed using RTPCR. Furthermore, both mtDNA depleted and non-depleted cells were characterised using immunocytochemistry to detect the expression of specific protein subunits of the OXPHOS system. Progressive mtDNA depletion was observed, using conventional PCR, in cells treated for 3 to 25 days with EthBr, while 42 days of culture resulted in complete depletion. RTPCR showed a progressive reduction followed by complete elimination of the mtDNA-encoded ND1, ND2, ND3, COX I and mtTFA transcripts. In addition, the expression of mtDNA-encoded protein subunits, e.g. COXI, of the OXPHOS system were reduced following mtDNA depletion whereas the expression of nuclear-DNA encoded protein subunits, e.g. COXVic, were unaltered. We hypothesize that the elimination of mtDNA and mtDNA transcripts from the donor cells will facilitate normal mtDNA replication and transcription in SCNT embryos, thus maintaining the strict unimaternal transmission of mtDNA to the offspring. Consequently, genetically identical offspring will be generated which have identical nuclear and mitochondrial DNA content, assuming oocytes from the same ovary are used. This technique is important for the generation of offspring for the livestock industry and animal models for the analysis of single gene disorders as well as the propagation of endangered species.