Transgene transmission in South American catfish (Rhamdia quelen) larvae by sperm-mediated gene transfer.
The silver catfish (Rhamdia quelen) is an endemic American fish species. The sperm of each species has its own peculiarities and biological characteristics, which influence the success of mass DNA transfer methods. Our objective in this study was to evaluate different sperm-mediated gene transfer (SMGT) methods to obtain transgenic silver catfish. Different treatments for the incorporation of a foreign pEGFP plasmid group were used: (1) dehydrated/rehydrated (DR), (2) dehydrated/rehydrated/electroporated (DRE), (3) electroporated (E), (4) incubated with seminal plasma (INC); and (5) incubated in the absence of seminal plasma (INCSP). Sperm motility, time of activity duration (TAD), fertilization rate (FR), hatching rate (HR) and sperm morphology were also evaluated. The polymerase chain reaction (PCR) positivity rates for the presence of the transgene were: DRE 60%; DR 40%; E 25%; INC 5% and INCSP 25%. The rates of embryo EGFP expression were: DRE 63%; DR 44%; E 34%; INC 8% and INCSP 38%. The fertilization rate in the control and DRE treatments groups were higher than in the DR group, but the E,INC and INCSP treatment groups had the lowest rate. The hatching rates of the DRE, DR and control groups were higher than in the INCSP, INC and E treatment groups (P>0.05). There were no differences among the DRE and DR, E and DR, E and INCSP groups in expression and PCR positivity rates of enhanced green fluorescent protein (EGFP) in embryos. Scanning electron microscopy also did not show any change in sperm morphology among treatment groups. To the best of our knowledge, this is the first report on transgene transmission of exogenous DNA into silver catfish larvae through SMGT technology.
Collares
Tiago
T
Biotechnology Center, Federal University of Pelotas, University Campus, CEP 96010-900, Pelotas, RS, Brazil. collares.t@gmail.com
Campos
Vinicius Farias
VF
Seixas
Fabiana Kommling
FK
Cavalcanti
Paulo V
PV
Dellagostin
Odir A
OA
Moreira
Heden Luiz M
HL
Deschamps
Joao Carlos
JC
D000818:Animals; D002397:Catfishes; D018274:Electroporation / Q000379:methods; D005260:Female; D005306:Fertilization; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008297:Male; D008855:Microscopy, Electron, Scanning / Q000379:methods; D016133:Polymerase Chain Reaction; D012098:Reproduction; D013094:Spermatozoa / Q000378:metabolism*; D019076:Transgenes
D049452:Green Fluorescent Proteins