Fate tracing of neurogenin2-expressing cells in the mouse retina using CreER™: LacZ.
Delineating the final fate of progenitor cells that transiently express a regulatory gene may shed light on how the gene participates in regulating retinal development. We describe the steps in tracing final fates of progenitor cells that once transiently express neurogenin2 (ngn2) during mouse retinal development with the binary, conditional Ngn2-CreER(™)-LacZ reporter system. Ngn2-CreER(™) mice (Zirlinger et al. Proc Natl Acad Sci USA 99:8084-8089, 2002), in which ngn2 promoter drives the expression of Cre-estrogen receptor CreER(™) (Littlewood et al. Nuc Acid Res 23:1686-1690, 1995; Hayashi and McMahon Dev Biol 244:305-318, 2002), are crossed with Rosa26-LoxP-LacZ reporter mice (Soriano Nat Genet 21:70-71, 1999), in which the expression of lacZ requires the removal of "stop" by Cre recombinase (Wagner et al. Transgenic Res 10:545-553, 2001). 4-hydroxytamoxifen (4-OHT), a synthetic ligand with high affinity for ER(™), is administered to double transgenic embryos and/or neonatal mice. Binding of 4-OHT to Cre-ER(™) activates Cre recombinase, which then catalyzes the removal of the "stop" sequence from the LoxP-LacZ transgene, leading to lacZ expression in cells that express ngn2. Retinal tissues are fixed at different time points after 4-OHT treatment and analyzed for LacZ activities by colorimetric reaction. Double-labeling with a cell type-specific marker can be used to define the identity of a LacZ(+) cell. Combining persisted lacZ expression through the life of the cell and the short half-life (0.5-2 h) of 4-OHT (Danielian et al. Curr Biol 8:1323-1326, 1998), this system offers the opportunity to track the final fates of cells that have expressed ngn2 during the brief presence of 4-OHT administered during retinal development.
National Eye Institute, National Institutes of Health, Bethesda, MD, USA. mawenxin@nei.nih.gov
Wenxin
W
Ma
Shu-Zhen
SZ
Wang
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D001947:Breeding; D018507:Gene Expression Regulation, Developmental; D060005:Genotyping Techniques; D059765:Homologous Recombination; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D009419:Nerve Tissue Proteins / Q000235:genetics*; D012160:Retina / Q000166:cytology / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D019426:Integrases; D009419:Nerve Tissue Proteins; C102938:Neurog2 protein, mouse; C045073:Cre recombinase