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Title: DNA PREPARATION METHOD CAN INFLUENCE OUTCOME OF TRANSGENIC ANIMAL EXPERIMENTS.

Author
item Wall, Robert
item PALEYANDA, REHKA - RED CROSS
item Foster Frey, Juli
item Powell, Anne
item REXROAD, JR., CAIRD - 1265-45-00
item LUBON, HENRY - RED CROSS

Submitted to: Animal Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/30/1999
Publication Date: 6/1/2004
Citation: Wall, R.J., Paleyanda, R., Foster Frey, J.A., Powell, A.M., Rexroad, Jr., C.E., Lubon, H. 2000. DNA preparation method can influence outcome of animal experiments. Animal Biotechnology Journal. 11:19-32.

Interpretive Summary: The efficiency of producing transgenic animals is low and as a consequence the cost of production are high. There are three major causes of the low efficiency: embryo survival, rate of introduction of the new gene into an animals genome and lack of proper function of the new gene. One of the things that could have a detrimental influence on embryo survival is contaminating compounds co-purified during the preparation of the genes. This study was designed to compare a standard DNA preparation method, Gel purification, that has the potential of introducing unknown impurities with a DNA perpetration method, sodium chloride gradient, that is far less likely to contribute any impurities during the DNA purification process. These two methods were tested during the production of transgenic mice and sheep. It was found that embryo survival was almost doubled in both sheep and mice if the sodium chloride gradient purification method was used. However, the proportion of transgenic animals produced, based either on embryos used or offspring born, was not different for either of the purification methods. These results suggest that, as we hypothesized, the DNA prepared by Gel purification methods may allow a slightly embryo toxic compound to be co-purified with the DNA.

Technical Abstract: In our continuing quest to improve the efficiency of producing transgenic animals, we have compared the influence of two transgene purification techniques on the efficiency of creating transgenic sheep and mice. Three hundred and eighty seven sheep zygotes and 2,737 mouse zygotes were microinjected with one of four transgenes. Transgenes were isolated from plasmid sequences either by agarose gel electrophoresis followed by gel extraction or by a single step sodium chloride gradient fractionation technique. Four transgenic sheep and 61 transgenic mice were produced. Both sheep and mice embryos responded similarly to transgene preparation methods. Overall, pregnancy rate was higher for recipients that received embryos injected with NaCl purified DNA (mean ? SEM: 64 +/ 7% vs. 38 +/ 7%). Furthermore, offspring per zygote transferred (NaCl, 22 +/ 3% vs. Gel, 12 +/ 3%) and transgenics born per zygote transferred (NaCl, 3.9 +/ 0.6% vs. Gel, 1.5 +/ 0.6%) were higher when the NaCl purified DNA was used. However, the proportion of offspring born that were identified as transgenic did not differ between transgene purification methods. Transgenes responded differently to methods of preparation. One of the four genes yielded a significantly higher proportion of transgenics when the transgene was prepared by NaCl purification. These data suggest that on average the NaCl gradient purification technique results in a higher embryo survival rate to term for both sheep and mice, but the technique has no influence on rate of transgene integration.