Burgess, S., and Lin, S.
ABSTRACT
Protocol Summary:
Moloney Murine Leukemia Virus (MLV) core particles were produced in a GT186 packaging cell line (1) (Genbank accession: JN244738.1) and pseudotyped with Vesicular Stomatitis Virus (VSV) envelope (G) protein. The virus was super-concentrated by high-speed centrifugation and resuspended in PBS. Virus was injected into T/AB-5 embryos at the 1-4000 cell stage (approximately 3hpf). Embryos were heat shocked at 37°C for 1.5 hours and then raised by normal animal husbandry to sexual maturity. Mature injected adults were either inbred or outcrossed to generate heterozygous transgenic male fish. The F1 males were raised to sexual maturity and then the sperm samples were cryopreserved; simultaneously genomic DNA was isolated from the tailfin and the genomic locations of the retroviral integrations was identified by a combination of linker-mediated PCR and Illumina paired-end sequencing.