Transgenic Zebrafish for Lineage Tracing

Tg(kdrl:HsHRAS-mCherry)^s896 animals (referred to as kdrl:memCherry for clarity) were previously described11 (link). Tg(kdrl:Cre)^S898 and Tg(βactin2:loxP-STOP-loxP-DsRed-express)^sd5 adults were mated, and their progeny screened for the presence of DsRed⁺ vasculature at 48 hpf. Positive embryos were raised to adulthood; some were sacrificed to analyze WKM at several ages (7 weeks to 6 months) by flow cytometry. Flow cytometry was performed as described9 (link), and sytox was used as a vital dye to exclude dead cells. Imaging was performed on an SP5 deconvolution confocal microscope (Leica, Germany). For time-lapse imaging, double transgenic Tg(kdrl:HsHRAS-mCherry)^s896; Tg(cmyb:eGFP) embryos were first screened for fluorescence, then anesthetized in tricaine and embedded in agarose. Time-lapse imaging was usually performed between 22 hpf and 36 hpf, in an environmental chamber maintained at 28°C. Raw data was analyzed using Volocity software (Improvision, Lexington, MA), and exported in Quicktime format.

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Bertrand J.Y., Chi N.C., Santoso B., Teng S., Stainier D.Y, & Traver D. (2010). Hematopoietic stem cells derive directly from aortic endothelium during development. Nature, 464(7285), 108-111.

Publication 2009

Adults Agarose Animals Cells Confocal microscope Embryos Flow cytometry Fluorescence Transgenic Tricaine

Corresponding Organization :

Other organizations : University of California, San Diego, University of California, San Francisco

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Variable analysis

independent variables

Presence or absence of the Tg(kdrl:Cre)S898 and Tg(βactin2:loxP-STOP-loxP-DsRed-express)sd5 transgenes

dependent variables

Presence of DsRed+ vasculature at 48 hpf
Characteristics of the whole kidney marrow (WKM) analyzed by flow cytometry at different ages (7 weeks to 6 months)

control variables

Maintenance of embryos at 28°C during time-lapse imaging
Use of sytox as a vital dye to exclude dead cells during flow cytometry

controls

Positive control: Tg(kdrl:HsHRAS-mCherry)s896 (kdrl:memCherry) animals previously described
Negative control: Not explicitly mentioned

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