Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fibroblast cell nuclei

Adult animal cloning has progressed to allow the production of offspring cl oned from adult cells. however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabol ic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofruct okinase (PFK), to determine if their detection in nuclear transfer (NT) emb ryos mimics that determined for in vitro produced embryos. A day 40 nuclear transfer produced fetus derived from an adult cell line was collected and fetal fibroblast cultures were established and maintained. Reconstructed NT embryos were then produced from this cell line, and RT-PCR was used to eva luate mRNA reprogramming. All three mRNAs encoding these enzymes were detec ted in the regenerated fetal fibroblast cell line. Detection patterns were first determined for IVF produced embryos (1-cell, 2-cell, 6-8 cell, morula , and blastocyst stages) to compare with their detection in NT embryos. PFK has three subunits: PFK-L, PFK-M, and PFK-P. PFK-L and PFK-P were not dete cted in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrate d that PFK was not detected in 8-cell NT embryos but was detected in NT bla stocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastoc yst stage in both bovine IVF and NT embryos. LDH A and LDH-B were detected in bovine oocytes and in all stages of IVF and NT embryos examined up to th e blastocyst stage. A third subunit, LDH-C was not detected at the blastocy st stage in IVF or NT embryos but was detected in all earlier stages and in mature oocytes. In addition, LDH-C mRNA was detected in gonad isolated fro m the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and theref ore must be properly reprogrammed following nuclear transfer. (C) 2000 Wile y-Liss, Inc.