Journal: microPublication Biology
Article Title: Generation of six4 -nlsRFP: a red somatic gonadal nuclear marker for live imaging Drosophila gonadogenesis
doi: 10.17912/micropub.biology.001652
Figure Lengend Snippet: (A) LR clonase reaction used to insert the Six4 enhancer sequence upstream of the nuclear red fluorescent protein sequence, mCherry:Renilla(nls), in the Vanglow#83339 plasmid. Entry clone and toxic byproduct plasmids are Kanamycin resistant (KanR), and destination vector and expression clone are Ampicillin resistant (AmpR). The construct injected is labeled as expression clone. (B) Sequence map of the plasmid generated to develop the six4 nlsRFP reporter. The Six4 enhancer noted on the map is the third intron of the D- Six4 gene sequence, diagrammed below the plasmid (exons, orange; UTRs, gray; introns, horizontal line). (C-H) Whole embryo, fixed tissue immunostaining for RFP to show six4 nlsRFP reporter expression for the construct injected on chromosomes III (C-E) and II (F-H) at embryonic stages 11 (C,F) , 15-16 (D,G) , and 17 (E,H) with the gonad outlined by yellow boxes. Expression of Six4 in the ventral nervous system (H, yellow arrow). Prime panels show high magnification of gonads in corresponding panels, outlined with dotted white line. RFP, red; Fas3, white. ( I-M ) Live imaging of stem cell niche assembly during embryonic stages 15-17 using the six4 nlsRFP reporter (magenta) to show SGP nuclei. Two individual niche cells over time (I-I'''', yellow arrows). Gonad outlined in white. Niche within gonad in I'''' also outlined in white. Time indicated in hh:mm:ss.
Article Snippet: An LR clonase reaction was performed to induce recombination at the attL and attR sites between these plasmids (ThermoFisher Scientific).
Techniques: Sequencing, Plasmid Preparation, Expressing, Construct, Injection, Labeling, Generated, Immunostaining, Imaging
Addgene inc is a verified supplier 
