Figure 1.

Restoration of Lztfl1 expression in Lztfl1^gt/gt mice by tamoxifen (TMX)-inducible FLP recombinase. (A) Schematic of the wild-type, gene-trap (gt) mutant and rescued alleles of Lztfl1. Black boxes represent Lztfl1 exons. Exon numbers are described above the black boxes. TMX administration induces excision of the gene-trap by FLP recombinase and converts the gene-trap allele into a functionally normal wild-type allele. SA, splice acceptor site; IRES, internal ribosome entry site; LacZ, β-galactosidase reporter; pA, poly(A) signal; PGK, mouse phosphoglycerate kinase 1 promoter; Neo, neomycin resistance gene. (B and C) Validation of the gene-trap cassette excision after TMX injection (at 1 week post-TMX injection). (B) Genomic DNAs from mouse tail snips were isolated before and after TMX injection, and excision of the gene-trap was assessed by PCR. (C) Genomic DNAs were extracted from the outer nuclear layer (ONL) of retinal sections by laser-capture microdissection, and excision of the gene-trap was analyzed by PCR. All mice used are Lztfl1^gt/gt, and FlpER genotypes are indicated (FLP⁻ or FLP⁺). (D) Restoration of Lztfl1 expression at the mRNA level (at 1 week post-TMX injection). Total RNA was extracted from the whole eye with or without TMX injection, and the amount of Lztfl1 mRNA was assessed by qRT-PCR assays. Primers specific to Lztfl1 exons 2 and 10 were used (n = 3 for each group). Error bars represent the standard error of the mean. (E) Restoration of Lztfl1 expression at the protein level. Lztfl1^gt/gt;FLP⁺ mice were injected with TMX at P30–34, and eyes were collected 1 week (w), 2 weeks and 3 weeks later for immunoblotting. Lztfl1^+/gt;FLP⁺ and uninjected Lztfl1^gt/gt;FLP⁺ mice were used as wild-type and mutant controls, respectively. Each lane represents an individual animal. β-actin was used as a loading control.