Evaluation of salmon sperm electroporation for gene transfer
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The aim of this thesis was to evaluate the potential of using sperm cells as a vector for gene transfer in chinook salmon (Oncorhynchus tshawytscha). Preliminary studies indicated that while salmon sperm were inefficient at picking up DNA during simple incubation, electroporation of the sperm significantly enhanced the sperm/DNA association. Therefore, a number of factors were examined to identify the optimal conditions for DNA uptake by salmon sperm and the subsequent transfer of foreign DNA into salmon embryos through fertilisation. The effect of electroporation on salmon sperm/DNA association was examined using bacterial transformation, the polymerase chain reaction, dot blot and Southern analysis. Sperm were electroporated in the presence of circular or linear pRSV–lacZ DNA at 20 µg/ml and 100 µg/ml, with field strengths of 625 V/cm or 1000 V/cm, a pulse length of 27.4 ms, and with either a single or double pulse. Electroporation enhanced sperm retention of circular and linear pRSV-lacZ DNA, and retention was greatest at the higher field strength with two pulses, and a DNA concentration of 100 µg/ml. The sperm/DNA association was strong as it resisted repeated washing and digestion by DNase 1, suggesting that some of the foreign DNA was internalised into the sperm. It was estimated that, following washing and DNase 1 digestion, on average, 0.12 fg (16 copies), of DNA were associated with each sperm cell. The ability of electroporated sperm to transfer plasmid DNA into embryos was investigated using a number of combinations of field strength, pulse length, pulse number and DNA concentration. Optimal conditions were found to be two pulses of 27.4 ms at 1000 V/cm, using 0.5 x HBS, with 200 µg/ml of plasmid DNA The transferred DNA was detected by PCR in up to 85% of the resulting 12 week old fry. Gene transfer efficiencies of up to 47% were repeatedly achieved over two separate spawning seasons. Southern analysis failed to detect any plasmid DNA in these fry, indicating that although electroporated sperm are capable of transferring the plasmid DNA, the levels of this DNA in the fry are low and the resulting fry are mosaic. PCR and Southern analysis of DNA extracted from embryos of five developmental stages was also carried out. Introduced plasmid DNA was detected in over 90% of the 0.8 day old embryos, and up to 107 copies of the plasmid DNA were observed in 1.2 day old embryos. The results indicated that the DNA introduced by electroporated sperm is amplified during the early stages of embryo development, remains predominantly extrachromosomal in an unmodified form, and is gradually lost during development. Expression of the introduced pRSV-lacZ DNA was detected in only a small number of embryos despite the presence of high levels of plasmid DNA in most of the embryos analysed. The methods of sperm electroporation and egg microinjection for gene transfer were directly compared. In a parallel study, a growth hormone gene construct, OnMTGH1, was transferred into chinook and coho salmon using both these methods. While the microinjected DNA was converted into high molecular weight concatemers, the sperm transferred DNA remained unmodified after introduction into salmon embryos. A greater proportion of 11 day old embryos resulting from electroporated sperm (5/5) were found to contain OnMTGH1 DNA than those resulting from microinjected eggs (4/8). However, no introduced DNA, or growth enhanced individuals, were detected in the 10 month old salmon developing from sperm electroporated with the GH construct. In contrast, introduced DNA was detected in 12% of the microinjected group, and growth enhanced individuals were obtained. It is clear from these results that electroporated salmon sperm can successfully take up DNA, and are efficient vectors for transferring genes into embryos. However, the introduced DNA does not persist and the levels decrease as the fish develop. Nevertheless, sperm electroporation has the potential to be further developed as an efficient mass gene transfer method in salmon.