Transfection Reagent-Mediated Gene Transfer For The Pacific White Shrimp Litopenaeus Vannamei

Abstract

Transfection reagents facilitate foreign DNA entry into cells, and thus provide an alternative to other gene transfer procedures available for shrimp. This study explored the application of four commercially available transfection reagents (SuperFect, Effectene, Lipofectamine 2000, and JetPEI) to carry a partial sequence of the Taura Syndrome Virus coat protein (TSV-CP) into Litopenaeus vannamei zygotes and Artemia franciscana eggs. Suitable reagents were selected based on shrimp hatching, transient gene expression via reverse transcription-polymerase chain reaction (RT-PCR), or transgene detection via polymerase chain reaction (PCR). The percentage of hatched nauplii was not significantly different among treatments in shrimp or Artemia, however, reduced hatching was observed in shrimp exposed to DNA/Lipofectamine 2000 compared to the mock-treated shrimp and shrimp exposed to DNA alone. Further data analysis showed that the manipulation of shrimp eggs prior to the formation of the hatching envelope (up to 16 minutes post-spawning), yielded poor nauplii hatching (percentage of hatched nauplii in mock-treated eggs = 5.8 %), as zygotes were more sensitive to the experimental procedure. In addition, the percentage of hatched nauplii increased significantly as shrimp zygotes were manipulated after the hatching envelope was developed (approximately 17-55 minutes post-spawning, mean % hatching = 46 %). TSV-CP expression was detected in nine-day old shrimp that were previously exposed to DNA alone, Effectene, and jetPEI/DNA complexes during the one-cell stage, and in Artemia transfected with Effectene and jetPEI DNA complexes, but not with DNA alone. TSV-CP expression was detected in shrimp transfected in the presence of jetPEI up to 191 days post-spawning. In an effort to study the green fluorescent protein (GFP) as a reporter gene for the shrimp system, both shrimp sperm cells and Artemia eggs were transfected with a GFP construct. Endogenous fluorescence was present in both the Artemia and shrimp sperm cells which made it difficult to detect GFP using either confocal or epifluorescence microscopy. However, Artemia that were previously transfected with the GFP exhibited higher fluorescence in the cells lining the end of the midgut just prior to the hindgut joint. In addition, Artemia that were previously transfected with GFP and jetPEI also exhibited fluorescence in the wall of the gastric caeca organ. Although the GFP gene and its expression were detected in Artemia via PCR and RT-PCR, this reporter gene may not be suitable for easy screening of transgenic shrimp due to the shrimp's endogenous fluorescence. In conclusion, this study indicates that both Effectene and JetPEI can successfully deliver foreign DNA into shrimp zygotes and Artemia eggs, although, jetPEI's transfection capabilities are uninhibited by the presence of the hatching envelope in shrimp.