turn signals body musculature to grow. Because of its close relationship with growth, IGF-I is gaining increasing acceptance as an indicator of growth rate (Fruchtman et al., 1997). Rapid detection of IGF-I could enable experimenters to screen combinations of variables (temperature, feed content and delivery method, and dissolved oxygen content of the water, for example) that would be exceedingly difficult or practically impossible on a farm-trial scale, since growth rate could be measured without the extensive commitment of resources needed for replicate trials on a large scale. Our efforts to develop, validate, and begin to apply an effective means of detecting IGF-I are closer to the outset than they are to completion, but we have passed some important milestones.
Methods and Materials
The first objective was to clone the tilapia (O. niloticus) IGF-I gene. Liver was collected from freshwater O. niloticus grown at Florida International University, frozen immediately in liquid nitrogen, and stored at -80” C. The frozen tissues were transported to North Carolina State University (NCSU) for processing in Dr. R. BorskiÕs laboratory, with the participation of personnel from both laboratories.
Total RNA was isolated using the guanidium thiocyanate procedure as described elsewhere. Liver total RNA was further extracted with lithium chloride to remove excess glycogen and other potentially interfering compounds. Oligonucleotide primers based on O. mossambicus IGF-I were used to clone the gene in O. niloticus. The outer primers span from within the 5' promoter region (forward: 5'-TTCTCCAAAACGAGCCTGCG-3') into the E domain (reverse: 5'-TCTGCTACTAACCTTGGGTGC-3'). First strand cDNA was synthesized from 1 µg total RNA using hot start Taq polymerase commercial reagents (Omniscript, Qiagen) in a total reaction volume of 20 µl. Extension was performed for 60 min at 37oC. Termination of the reaction occurred by incubation at 94oC for 5 min.
The minute quantity of cDNA obtained by means of the process outlined above was amplified using the Polymerase Chain Reaction (PCR). A 3 µl sample of the reverse transcription reaction was combined with IGF-I specific primers. The PCR was performed for 30 cycles, each consisting of denaturation at 94oC for 1 min, annealing at 56oC for
1 min, and extension at 72oC for 90 sec, followed by a final extension at 72oC for 15 min. PCR products were purified (Qiagen, Valencia, CA) and ligated and cloned into pCRII vector (Invitrogen, San Diego, CA). The final product for each gene was sequenced in the forward and reverse directions (NCSU DNA Sequencing & Mapping Facility
and University of Chicago Cancer Research Center DNA Sequencing Facility) and compared with known IGF-I sequences using BLAST (Basic Local Alignment Search Tool; National Center for Biotechnology Information).
A single experiment was run in order to carry out a preliminary test of the capability of the IGF-I cDNA to be used as a biological indicator of growth rate in tilapia. For this study, tilapia were grown at Florida International University under three feeding rations. In the preliminary study, Northern Blot analyses were used to determine whether the IGF-I gene expression (mRNA) is elevated in the livers of faster versus slower growing animals. To obtain differential growth in a minimal amount of time, fish were held for one month on either a maintenance feed ration, or on a near-satiation feed ration. Liver samples were collected after one month, frozen on dry ice, and stored at -80 oC until analyzed by the Northern Blot technique.
Total RNA from liver of normal and slow growing O. niloticus was extracted with Trizol reagent and quantified by spectrophotometry at 260 nm. Ten micrograms of sample RNA was run on a 1% formaldehyde agarose gel with buffers and protocols provided in the Northern Max kit (Ambion). RNA was blotted onto positively-charged nylon membranes by upward transfer and then fixed to the membrane by UV crosslinking. Blots were prehybridized at 42oC for two hours prior to hybridizing overnight using a 32P-CTP labeled
IGF-I cDNA. After washing, membranes were exposed for 48 hours using a quantitative phosphorimaging method.
In addition, determinations of total RNA, total DNA, and the RNA:DNA ratio were carried out using a spectrophotometer during the course of nucleotide extraction for Northern Blot analysis.
Results
The O. niloticus IGF cDNA corresponds to a 539 bp fragment of the prepro IGF-gene that spans from within the 5' promoter region into the E domain. The deduced amino acid sequence of O. niloticus cDNA was compared with known sequences in other vertebrate species. Identities between O. niloticus IGF-I and the corresponding region in fish species including O. mossambicus ranged from 75Š99%, whereas those between O. niloticus IGF-I and nonfish species ranged from around 70Š80%. The cDNA encoded a mature IGF-I peptide (consisting of B, C, A, and
Table 1. Growth, RNA, DNA, and RNA/DNA ratio data, Experiment 1.