American Society of Hematology

Figure 1.

$Figure 1. In vivo competitive gain-of-function screen selects for genes promoting repopulation latency. (A) Screen outline: From 64 candidate genes obtained by comparative in silico analysis, lentiviral vectors were produced individually and used at matching multiplicity of infection (MOI) to transduce CB lineage negative (Lin−) CD34+CD38− cells for a stem screen and CB Lin−CD34+CD38+ for a progenitor screen. The next day, 16 pools were assembled, each from 8 individually transduced cell populations, and transplanted intrafemoral into 5 NSG or 4 NSG-SGM3 mice per pool, respectively. Aliquots of individual and pooled samples were in vitro cultured for an additional 5 days for flow cytometric (BFP/gene marking) analysis and genomic DNA isolation for pool composition analysis at input. BM of mice was harvested at 20 or 4.5 weeks for flow cytometric and genomic DNA analysis, and engraftment and candidate gene contribution was scored. (B) For each candidate gene, a score was calculated in both screens by multiplying the output/input ratio for %BFP+ and percent vector copies (barcode abundance) per mouse, and adding a competition score in increments if the gene’s contribution to a pool was more than 1/8 (supplemental Figure 9A).The final score was obtained by accumulation of these individual scores/mouse for all mice analyzed carrying that candidate gene, and used to create a matrix of scores in both screens. Genes scoring in the stem screen only are highlighted in yellow. The score obtained for C3orf54 is highlighted in red. (C) The progeny of 10 000 CB Lin−CD34+CD38− cells transduced with the indicated overexpression vectors were transplanted into NSG mice 1 day after transduction. Input was assessed as mean of transduction efficiency at day 3 and day 6 posttransduction (ptd). Mice were analyzed for human (CD45+) chimerism (injected femur) and %BFP+ within the CD45+ cell fraction after 4 and 20 weeks. Shown are log2 ratios (%BFP+ of CD45+ in vivo output/%BFP+ in vitro input) of 2 to 5 independent experiments, adding up to 9 to 33 mice per time point and candidate. The table below indicates the P values (Student t test) and FC for the comparison of 4- and 20-week ratios per gene. *P < .05; **P < .01; ***P < .001. DIG, densely interconnected genes.$

In vivo competitive gain-of-function screen selects for genes promoting repopulation latency. (A) Screen outline: From 64 candidate genes obtained by comparative in silico analysis, lentiviral vectors were produced individually and used at matching multiplicity of infection (MOI) to transduce CB lineage negative (Lin⁻) CD34⁺CD38⁻ cells for a stem screen and CB Lin⁻CD34⁺CD38⁺ for a progenitor screen. The next day, 16 pools were assembled, each from 8 individually transduced cell populations, and transplanted intrafemoral into 5 NSG or 4 NSG-SGM3 mice per pool, respectively. Aliquots of individual and pooled samples were in vitro cultured for an additional 5 days for flow cytometric (BFP/gene marking) analysis and genomic DNA isolation for pool composition analysis at input. BM of mice was harvested at 20 or 4.5 weeks for flow cytometric and genomic DNA analysis, and engraftment and candidate gene contribution was scored. (B) For each candidate gene, a score was calculated in both screens by multiplying the output/input ratio for %BFP⁺ and percent vector copies (barcode abundance) per mouse, and adding a competition score in increments if the gene’s contribution to a pool was more than 1/8 (supplemental Figure 9A).The final score was obtained by accumulation of these individual scores/mouse for all mice analyzed carrying that candidate gene, and used to create a matrix of scores in both screens. Genes scoring in the stem screen only are highlighted in yellow. The score obtained for C3orf54 is highlighted in red. (C) The progeny of 10 000 CB Lin⁻CD34⁺CD38⁻ cells transduced with the indicated overexpression vectors were transplanted into NSG mice 1 day after transduction. Input was assessed as mean of transduction efficiency at day 3 and day 6 posttransduction (ptd). Mice were analyzed for human (CD45⁺) chimerism (injected femur) and %BFP⁺ within the CD45⁺ cell fraction after 4 and 20 weeks. Shown are log2 ratios (%BFP⁺ of CD45⁺ in vivo output/%BFP⁺ in vitro input) of 2 to 5 independent experiments, adding up to 9 to 33 mice per time point and candidate. The table below indicates the P values (Student t test) and FC for the comparison of 4- and 20-week ratios per gene. *P < .05; **P < .01; ***P < .001. DIG, densely interconnected genes.

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