Inactivation of Oct-1. (A) Schematic of the Oct-1 murine genomic locus and targeting vector. Exons are denoted by black boxes and positions of internal and external probes are indicated with triangles. Restriction enzymes: RI, EcoRI; H, HindIII; N, NheI; NT1, NotI. (B) Genomic blot by DNA derived from ES cells. The DNA was digested with NheI and probed with the 5′ external probe indicated in A. The endogenous allele is 6 kb and the targeted allele is 7.2 kb. Wt, wild-type allele; Mut, targeted allele. (C) Genotyping of tail DNA by PCR. PCR conditions and primers are described in Materials and Methods. Primers distinguished a 304-bp fragment of the targeted allele from a 424-bp fragment of the wild-type allele. (D) Reverse transcription-PCR of RNA from Oct-1 mutant mice. Primers amplified a region from exon 2 to the POU domain.

Oct-1^−/− immortalized cell lines fail to activate octamer-containing promoters. (A) H2B (black bars) and immunoglobulin V_H (white bars) promoter constructs were transfected. pGL3 vector was used to establish a baseline of firefly luciferase activity (gray bars). (B) Point mutation in the octamer element abolishes promoter activity. Levels of exogenous Oct-1 delivered via a retroviral vector are shown in Western blot (inset). Lane 1 represents a wild-type cell line, lane 2 depicts a mutant cell line infected with retrovirus carrying pBABE-hOct-1, and lane 3 denotes the same line carrying an empty pBABE vector. All cell lines used in this experiments were single-cell cloned. *, Oct-1 protein.

Oct-1-deficient embryos are runted and anemic and show reduced β-globin expression. (A) Comparison of an Oct-1-deficient embryo (right) with a wild-type littermate control (left) at E12.5. Oct-1 mutants appeared smaller with pale fetal livers that are reduced in size (arrows). (B and C) Flow cytometry analysis of TER-119-positive cells from E12.5 fetal livers. Oct-1 mutants exhibit a decreased proportion (B) and fewer total numbers (C) of TER-119-positive cells. Three wild-type and four homozygous Oct-1 mutant animals were used. Error bars denote standard deviations. (D) Fluorescence-activated cell sorting analysis of bone marrow from adoptive transfer of fetal livers. Irradiated recipient animals were injected with fetal liver cells from E12.5 Oct-1^−/− embryos. At 16 weeks after transfer, bone marrow cells from the recipient mice were analyzed by staining with antibodies directed against TER-119 and CD45.2/Ly-5.2. (E) Separation of fetal and adult globins by Triton-acetic acid-urea gel electrophoresis of protein extract from E11.5 embryos. Lane 1: peripheral blood control derived from 8-week-old adult mouse; lanes 2 to 8, embryos with their genotypes indicated. Carb. Anhyd., carbonic anhydrase. (F) Real-time PCR amplification curves for β-globin and β-actin. Data are shown for one representative set of experiments. (G) Quantitation of globin RNA levels by SYBR green real-time PCR. RNA was purified from E12.5 TER-119-positive fetal liver cells.

Characterization of Oct-1 protein levels in mutant cells. (A) DNA binding activity is decreased in both heterozygous and homozygous MEF nuclear extracts. Lanes contained 5 μg of nuclear extract. HeLa extract was included as a positive control (lane 2). Lanes 3 and 5 contained wild-type extract, lane 4 had extract from heterozygous MEFs, and, lane 6 had extract from Oct-1-deficient MEFs. Oct-1/DNA complex is indicated by an asterisk. (B) Oct-1 DNA binding activity is dramatically reduced in mutant nuclear extract. EMSA of nuclear extract derived from wild-type (lanes 3 to 5, 11) and Oct-1-deficient immortalized cell lines (lanes 6 to 8, 12). Lanes contained 10 μg of total protein. **, supershifted complex formed upon addition of anti-Oct-1 antibody (lanes 4 and 7). Reactions in lanes 1 to 8 were incubated with an octamer oligonucleotide. and reactions in lanes 9 to 12 were incubated with a mutant. (C and D) A 1-s exposure and 2-min exposure, respectively, of a Western blot with a combination of three anti-Oct-1 antibodies (Materials and Methods). The load consisted of 2.5 μg of nuclear extract (lanes 1 and 2), representing 1% of the input sample. Truncated polypeptides are indicated by an asterisk (lane 8). Nuclear extract load (L[NE]), flowthrough (FT), wash (0.2 M W), and elution (1.0 M).

Expression of putative Oct-1-dependent target genes is unaltered in the absence of Oct-1. Three independently isolated strains of Oct-1^−/− primary MEFs were tested. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were used as loading controls.