Fig 1.
The ES cell line XK097 contains a gene trap insertion in the Abcf1 gene.
A) The genomic and mRNA location of the Bay Genomics pGT0Lxf vector (gene trap) on chromosome 17 corresponding to the Abcf1 locus in the XK097 ES cell line. The locations and names of the primers used for screening are in round brackets. The size of the PCR product is indicated in square brackets. B) The ES cell line, XK097, contains the gene trap sequence between exon 7 and 8 in the Abcf1 gene. Genomic DNA analysis of the XK097 cell line (left panel) compared with a wild type ES cell line (right panel). “IB” (Int1-f and Bay cDNA (1250bp)) primer combination indicates the presence of the gene trap cassette in the Abcf1 locus. “-” is the negative water alone control and “+” is the positive control (Rps15 (350bp)) for the PCR. C) RT-PCR of the ES cell line, XK097, produces a transcript containing exon 7 and the Bay Genomics vector sequence. RT-PCR analysis of the XK097 cell line (Abcf1+/-; left) compared with a wild type ES cell line (Abcf1+/+; right). 7 = Ex7-f-Bay-cDNA primers, F = Ex7-Ex8 primers, β = β-actin primers.
Table 1.
Genotypes of the offspring from Abcf1+/- inter-crosses show that approximately 25% of blastocysts are Abcf1-/-.
Fig 2.
The Abcf1 promoter is highly expressed during early embryogenesis.
A) Blastocysts were isolated from 3.5 dpc female mice and stained with X-gal. Three separate phenotypes were observed: non-stained (Abcf1+/+), intermediate-stained (Abcf1+/-) and darkly stained (Abcf1-/-), which directly corresponded to their genotypes and to the gene copy number of the β-geo reporter. The darkly stained blastocysts also appeared to be slightly oblong in shape compared with the medium-stained embryos and lacked a zona pellucida. Images were taken using a 20× lens. B) To distinguish between homozygous-knockout and heterozygous blastocysts, the blastocysts from A were genotyped by PCR to confirm the genotypes. The top image shows the presence of the knockout allele in both blastocysts. The bottom image (taken using an QIAxcel Capillary Electrophoresis machine at Biomedical Research Centre, UBC) shows the presence of the wild type allele only in the heterozygous blastocyst.
Fig 3.
The Abcf1 promoter is highly expressed throughout embryogenesis.
A) 8.5 dpc and B) 11.5 dpc embryos were stained with X-gal, and Abcf1+/- heterozygous (blue) embryos showed strong staining throughout the embryo, compared with Abcf1+/+ wild type (white) littermate controls, which showed no staining. C) An 11.5 dpc embryo was sectioned and showed that the promoter was active in all tissues. Images were taken using a 3.5× objective.
Fig 4.
The Abcf1 promoter is active in whole adult mouse organs.
Whole organs were excised from Abcf1+/- mice and their Abcf1+/+ littermate controls and stained with X-gal for 24 h. There was also some endogenous X-gal staining in the wild-type liver, testis, and thymus, so these organs were stained for 4–12 h, depending on the development of endogenous expression in the wild-type control.
Fig 5.
X-gal expression is variable in tissues from Abcf1+/- adult mice.
Variable Abcf1 promoter activity was seen in A) Cerebral Cortex, B) Retina, C) Heart, D) Renal Cortex, E) Liver, F) Lung, G) Inguinal Lymph Node, H) Muscle, I) Pancreas, J) Skin, K) Small Intestine, L) Spleen, M) Thymus, and N) Testis. Tissues were stained with X-gal and counter-stained with nuclear fast red. Images were taken using the 40× objective.
Fig 6.
Quantitative representation of X-gal staining.
Image quantification was done using MatLab (MathWorks, Apple Hill Drive, MA, USA), as described in the material and methods section. 7 animals each of control (Abcf1+/+; no β-geo) and heterozygotes (Abcf1+/-; expressing β-geo) were sacrificed, tissues were extracted and staining intensities were measured. The error bar represents the standard deviation. Percentage 1–10% is characterized as light or faint staining, 10–40% as intermediate staining and greater than 40% as intense staining.