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FUNCTIONAL HAMMERHEAD RIBOZYMES AGAINST THE MOUSE TESTIS LACTATE DEHYDROGENASE.
Srinivasan, Shankar1, Ashtiani, Kourosh2, Goldberg, Erwin1, 1 2
ABSTRACT- Mammalian spermatogenesis is a highly complex process in which spermatogonial stem cells undergo differentiation over a period of weeks to months to generate mature spermatozoa. The success of this process is attributed mainly to the coordinated expression of a number of testis specific genes. One of the earliest genes to be expressed during this highly organized process is the testis specific isoform lactate dehydrogenase, (ldhc), an important glycolytic enzyme. Expression of ldhc mRNA begins in the preleptotene spermatocytes. One of the ways to elucidate the functional role of ldhc and establish its function during spermatogenesis is to knockout this gene. The presence of several repetitive sequences in introns of this gene and also the existence of ldh pesudogenes have been constant hurdles in using the conventional knockout procedures. An alternative strategy is to target degradation of ldhc mRNA with ribozymes. Ribozymes are catalytic RNA molecules that can be manipulated to bind and cleave specific RNA sequences. In recent years hammerhead ribozymes have been successful in the down regulation of several viral and oncogene RNA. Based on this approach five hammerhead ribozymes were designed to target 5 different regions of the open reading frame of the mouse ldhc (mldhc) mRNA. An in vitro cleavage assay showed that 3 of the 5 designed ribozymes cleaved this mRNA in a sequence and site specific manner. In vitro kinetics studies clearly showed a pattern typical of protein enzymes, viz. increased cleavage with increase in concentration and time. Moreover these ribozymes did not show much alteration in their cleavage kinetics between 37°C and 32°C. In vivo studies were done using these ribozymes by co-transfection of mouse fibroblast cells (NIH 3T3) and the human cervix cell line (HeLa) with mldhc cDNA and cDNA for each of the 3 ribozymes. Reverse transcription polymerase chain reaction (RTPCR) and western analysis clearly showed that these 3 ribozymes are equally active in vivo in down regulating mldhc expression. Mutation of an active nucleotide in the core of the hammerhead sequence abrogates the cleavage effect. These 3 ribozymes are being tested for their functional effects on mouse spermatogenesis by exogenous delivery and transgenic methods.
KEY WORDS: spermatogenesis, gene down regulation