Journal: bioRxiv

Article Title: Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts

doi: 10.1101/2020.04.16.045245

Figure Lengend Snippet: ( A ) Human lung adenocarcinoma (A549) cells were genetically engineered with the lineage tracing components by lentiviral transduction with (1) Luciferase-Neomycin R and antibiotic-selected, (2) Cas9-mCherry and fluorescence-sorted, (3) serial, high-titer TargetSite-GFP and fluorescence-sorted, and finally (4) triple-sgRNA BFP-Puromycin R and fluorescence-sorted, thus producing lineage tracing-competent A549-LT cells. ( B ) Serial, high-titer TargetSite-GFP lentiviral transduction strategy to achieve high copy-number. ( C ) Cells with high-shifted GFP fluorescence after successive TargetSite-GFP infections, indicating increasing copy-number of the Target Site. ( D ) Sample of 5,000 A549-LT cells prior to injection to estimate initial clonal diversity. Shown is a heat-map of the fraction of shared intBCs in all cell–cell comparisons. On average, approximately 22,000 unique, high-quality intBCs were identified per random sample of 5,000 cells; thus, we estimate approximately 2,150 distinct clones per 5,000 cells (assuming 10.3 intBCs per clonal population; ) at the beginning of the experiment. ( E ) Comparison of the size of each clonal population observed in mouse M5k in a pre-implantation sample of 5,000 cells ( in vitro ) and post-sacrifice ( in vivo ). There is no correlation between the in vitro and in vivo population sizes (Spearman’s ρ =-0.026).

Article Snippet: Guide RNAs for CRISPRi and CRISPRa experiments were chosen from the human CRISPRi/a v.2 libraries (Addgene #83969 and #83978, respectively) and were cloned into the pLG1 lentiviral backbone (Addgene #84832) using the annealing and ligation as previously described ( ).

Techniques: Transduction, Luciferase, Fluorescence, Injection, Clone Assay, Comparison, In Vitro, In Vivo