Germline Transgenesis and Insertional Mutagenesis in Schistosoma mansoni Mediated by Murine Leukemia Virus
Figure 2
Construction of Illumina libraries from virion transduced schistosomes.
A: Schematic representation of a representative MLV retrovirus construct integrated into the gDNA isolated from MLV-transduced organisms. The retrovirus cassette included the firefly luciferase reporter gene (LUC) driven by the S. mansoni actin 1.1 gene promoter (SmACT), and flanked by the 5′- and 3′-long terminal inverted repeats of the murine leukemia virus (LTR). The cassette also included the gene endowing neomycin resistance (neoR) and the psi motif (ψ) involved in packaging the viral DNA; B: Mechanical fragmentation of the genomic DNA; C: Repair of the fragment ends, adenylation, ligation of the Illumina adapters, and two rounds of semi-nested PCR (colored primers represent the sequencing primers); D: Size selection of the 5′-end (5′) and 3′-end (3′) libraries and gel extraction. The fragment selected from 300 bp to 500 bp (i) was employed to generate the libraries. A higher fragment (ii) was purified and stored as back up. The gel extracted and purified libraries were quantified by qPCR and loaded into Illumina flowcells. L, molecular size standards (ladder).