To date, there are no reports of established suitable RG or set of genes for expression studies in C. batrachus for any physiological conditions.
For expression studies, we generated a C-terminally myc-tagged SpARC1 construct.
For expression studies, we purified islets [49], and extracted total RNA using RNeasy (Qiagen).
For expression studies, animal intestines were flushed with cold PBS and either homogenized whole or scraped to enrich for enterocytes.
For expression studies, mosquito infection with P. falciparum was initially performed in Senegal, using blood of gametocyte carrier volunteers, as described [34].
For expression studies, E2f4 −/− homozygous and E2f4 +/− heterozygous knock-out mice were derived in a B6/C57 and 129S2/SvPas cross background [16], [31].
For expression studies, the gene was excised out by using NdeI and XhoI restriction enzymes and was cloned into pET28c at the same sites resulting in pET28c.fad.
For expression studies, the mutated insert was excised out of plit38.fad and cloned into pET28c by using the same cloning strategy as described above.
We amplified fragments containing miR-499 from genomic DNA using high-fidelity PCR (PlatinumTaq, Invitrogen) and directional cloning into pcDNA3.1-TOPO (Invitrogen) for expression studies in cell culture.
For expression studies, papl-1::apl-1 gfp apl-1 gfp was co-ing/µled wash the marker construct pRF4 (75 ng/µl), which contains rol-6(su1006).
The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans.
