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 cloned from adult cells, however many cloned calves die prenatally or shortly after birth. This study examined the expression of three important metabolic enzymes, lactate dehydrogenase (LDH), citrate synthase, and phosphofructokinase (PFK), to determine if their detection in nuclear transfer (NT) embryos 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 evaluate mRNA reprogramming. All three mRNAs encoding these enzymes were detected 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 detected in bovine oocytes. PFK subunits were not detected in 6-8 cell embryos but were detected in blastocysts. Results from NT embryo RT-PCR demonstrated that PFK was not detected in 8-cell NT embryos but was detected in NT blastocysts indicating that proper nuclear reprogramming had occurred. Citrate synthase was detected in oocytes and throughout development to the blastocyst 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 the blastocyst stage. A third subunit, LDH-C was not detected at the blastocyst 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 from the NT and an in vivo produced control fetus. These results indicate that the three metabolic enzymes maintain normal expression patterns and therefore must be properly reprogrammed following nuclear transfer.
Winger
Q A
QA
Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A&M University, College Station 77843-4466, USA.
Hill
J R
JR
Shin
T
T
Watson
A J
AJ
Kraemer
D C
DC
Westhusin
M E
ME
D000818:Animals; D001755:Blastocyst / Q000201:enzymology*; D002417:Cattle; D002460:Cell Line; D002478:Cells, Cultured; D002950:Citrate (si)-Synthase / Q000235:genetics* / Q000378:metabolism; D002999:Clone Cells; D017931:DNA Primers / Q000737:chemistry; D004624:Embryo Transfer; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D009865:Oocytes / Q000201:enzymology; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D012333:RNA, Messenger; D007770:L-Lactate Dehydrogenase; D002950:Citrate (si)-Synthase; D010732:Phosphofructokinase-1