Airway epithelial progenitors were isolated from adult tracheas of wild type mice
or E2f4 mouse mutants and cultured using well-established protocol (see
Methods)41 (link)42 (link). For the functional analysis of E2f4 in ALI
cultures we used adult E2f4+/+,
E2f4+/f;
R26CreERT2/LSLZsGreen1or E2f4f/f;
R26CreERT2/LSLZsGreen1 mice, as well as mice
lacking the R26LSLZsGreen1 allele. Briefly, after treatment with
0.5% pronase overnight, cells were cultured on collagen1-coated Transwell
dishes (Corning) under submerged conditions in media that allowed expansion of
airway progenitors42 (link) until confluence (7 days). The ALI was
established by removing media from the upper chamber of Transwell (Day 0 ALI)
and culturing cells in differentiation media (mTEC/serum free, RA media)42 (link) up to 8 days (Day 8 ALI). Treatment with 1 μM
4-hydroxytamoxifen (Tm) from day −5 to day 0 was used to induce Cre
mediated recombination36 (link). The efficiency of recombination was
analysed by qPCR, IF for E2f4 and expression of ZsGreen1, where appropriate.
or E2f4 mouse mutants and cultured using well-established protocol (see
Methods)41 (link)42 (link). For the functional analysis of E2f4 in ALI
cultures we used adult E2f4+/+,
E2f4+/f;
R26CreERT2/LSLZsGreen1or E2f4f/f;
R26CreERT2/LSLZsGreen1 mice, as well as mice
lacking the R26LSLZsGreen1 allele. Briefly, after treatment with
0.5% pronase overnight, cells were cultured on collagen1-coated Transwell
dishes (Corning) under submerged conditions in media that allowed expansion of
airway progenitors42 (link) until confluence (7 days). The ALI was
established by removing media from the upper chamber of Transwell (Day 0 ALI)
and culturing cells in differentiation media (mTEC/serum free, RA media)42 (link) up to 8 days (Day 8 ALI). Treatment with 1 μM
4-hydroxytamoxifen (Tm) from day −5 to day 0 was used to induce Cre
mediated recombination36 (link). The efficiency of recombination was
analysed by qPCR, IF for E2f4 and expression of ZsGreen1, where appropriate.
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