Development of retroviral vectors for insertional mutagenesis in XXXX haploid cells.

@entity1 @entity778 @entity74 @entity7021
UNASSIGNED: Insertional mutagenesis (IM) by retrovirus (RV) is a high-throughput approach for interrogating gene functions in model species. Haploid cell provides a unique system for genetic screening by IM and prosperous progress has been achieved in @entity1 cells. However, little was known in lower vertebrate cells. Here, we report development of retroviral vectors (rvSAchCVgfp, rvSAchCVpf and rvSAchSTpf) and establishment of IM library in @entity7021 haploid cells. Each vector contains a modified @entity74 ( @entity74 ) cassette, which could extend the mutated cell population including @entity74 insertions not in-frame or in weakly expressed genes. Virus titration determined by flow cytometry showed that rvSAchSTpf possessed the highest supernatant virus titer (1.5*10(5)TU/ml) in @entity7021 haploid cell, while rvSAchCVpf produced the lowest titer (2.8*10(4)TU/ml). However, quantification of proviral DNAs in transduced cells by droplet digital PCR (ddPCR) demonstrated that the "real titer" may be similar among the three vectors. Furthermore, an IM library was established by FACS of haploid cells transduced with rvSAchCVgfp at a MOI of 0.1. A single copy RV integration in the majority of cells was confirmed by ddPCR in the library. Notably, there was a significant decrease of haploid cell percentage after FACS, suggesting potential trapping for survival/growth essential genes. Our results demonstrated successful development of retroviral vectors for IM in @entity7021 haploid cells, serving for haploid genetic screening of host factors for @entity778 and genes underlying certain cellular processes in fish model.
