Heterozygous deletion of Atbf1 by the Cre-loxP system in mice causes preweaning mortality

Abstract

ATBF1 is a large nuclear protein that contains multiple zinc-finger motifs and four homeodomains. In mammals, ATBF1 regulates differentiation, and its mutation and/or downregulation is involved in tumorigenesis in several organs. To gain more insight into the physiological functions of ATBF1, we generated and validated a conditional allele of mouse Atbf1 in which exons 7 and 8 were flanked by loxP sites (Atbf1(flox) ). Germline deletion of a single Atbf1 allele was achieved by breeding to EIIa-cre transgenic mice, and Atbf1 heterozygous mice displayed reduced body weight, preweaning mortality, increased cell proliferation, and attenuated cytokeratin 18 expression, indicating haploinsufficiency of Atbf1. Floxed Atbf1 mice will help us understand such biological processes as neuronal differentiation and tumorigenesis.

Figure 1. Generation of the floxed Atbf1 allele (Atbf1^flox) in mice

a. Design of cloning vectors and targeting strategies. Part of the Atbf1 gene (from exon 6 to exon 10) is shown at top (wildtype allele). LoxP and Frt sequences are marked by triangle and diamond-shaped boxes respectively. The targeting construct was generated by inserting three Atbf1 fragments (6.2 kb, 0.9 kb and 4.7 kb) into pFlexible. Two hybridization probes (P1 and P2) used in Southern blot analyses are indicated in the mutant allele. The PGK-puroΔtk selection cassette was deleted by mating Atbf1^PGK mice with ACT-FLPe mice to generate the Atbf1^flox allele. Digestion of targeted genomic DNA by Xba I yields a 10.9 kb fragment for the wildtype allele and an 8.4 kb fragment for the mutant allele when the 5′ probe (P1) is used, while digestion by Xho I yields 7.4 kb wildtype and 5.1 kb mutant bands when the 3′ probe (P2) is used. b. Southern blot analyses of wildtype (wt) and targeted (PGK) alleles in ES cells, showing expected mutant fragments for both 5′ and 3′ probes.

Figure 2. Excision of exons 7 and 8 of Atbf1 by the Cre recombinase

a. Schematic representation of Cre-mediated deletion of exons 7 and 8. LoxP and Frt sequences are marked by triangle and diamond-shaped boxes respectively. Primers F1 and R2 are indicated by arrow heads. The Atbf1^Δ7&8 deletion allele was induced by Cre from adenoviruses or EIIa-cre mice. b. Five MEF clones incubated with adenoviruses expressing GFP or Cre were genotyped by a PCR using primers F1 and R2, identifying three Atbf1 alleles: Atbf1^flox (1248 bp), Atbf1^wt (1071 bp) and Atbf1^Δ7&8 (289 bp). c. Detection of the wildtype (532 bp) and truncated (197 bp) Atbf1 mRNA by RT-PCR in MEFs with different status of Atbf1 deletion. d. Detection of Atbf1 protein in MEFs with different Atbf1 deletion status by immunoblotting. Molecular masses of protein standards are shown to the left of the gel.

Figure 3. Reduced body size and weight in mice with deletion of one Atbf1 allele

a. Representative genotyping PCR. Eight mice were examined for Atbf1, Cre, and Il2 (an internal control) genotypes. Three Atbf1 alleles (Atbf1^flox, Atbf1^wt and Atbf1^Δ7&8) were detected in a single PCR. Two mice with the Cre⁺/Atbf1^flox/wt genotype (#1 and #8) showed a mosaic genotype. M, 1 Kb plus DNA standard (Invitrogen). b. Mosaic genotypes in different organs. Nine organs from mouse #1 in panel 3a were examined for the Atbf1 genotype. B, brain; Lv, liver; Lg, lung; H, heart; I, intestine; K, kidney; Sp, spleen; St, stomach; T, tail. c. Representative images of 21-day old mice with the wildtype (wt/wt) or heterozygous deletion (wt/Δ7&8) of Atbf1. d. Weights of wildtype mice (wt/wt, filled circles) and heterozygous mice (wt/Δ7&8, open circles) aged at 13 to 29 days. Atbf1 heterozygous mice were significantly lighter than their wildtype littermates.

Figure 4. Reduced Atbf1 expression in mice with Atbf1 deletion

a. Knockout of Atbf1 reduced the expression of the wildtype Atbf1 mRNA in most organs of mice at postnatal day 1 (left) and day 21 (right), as detected by real-time RT-PCR. The ratio of Atbf1 to β-actin mRNA levels for wildtype mouse brain was set to 1, and that for other organs were adjusted accordingly. The reduction by about half is obvious in the brain (B), liver (Lv), lung (Lg), heart (H), kidney (K) and stomach (St) but not in the intestine (I) and spleen (Sp) in mice at 21-day-old (right). b. Detection of the truncated Atbf1 mRNA by real-time RT-PCR in the brain, liver, heart, intestine, kidney and spleen but not in the lung and stomach of the Atbf1 heterozygous mice. c. Molecular and histological analyses of mouse kidneys from both Atbf1 wildtype (wt/wt) and heterozygous (wt/Δ7&8) mice. HE, representative images from Hematoxylin and Eosen stained sections. IHC staining confirmed reduced Atbf1 expression, increased Ki-67 expression and attenuated CK18 expression in the Atbf1 heterozygous mice (wt/Δ7&8) when compared to wildtype mice (wt/wt). All scale bars are 100 μm.

Figure 4. Reduced Atbf1 expression in mice with Atbf1 deletion

a. Knockout of Atbf1 reduced the expression of the wildtype Atbf1 mRNA in most organs of mice at postnatal day 1 (left) and day 21 (right), as detected by real-time RT-PCR. The ratio of Atbf1 to β-actin mRNA levels for wildtype mouse brain was set to 1, and that for other organs were adjusted accordingly. The reduction by about half is obvious in the brain (B), liver (Lv), lung (Lg), heart (H), kidney (K) and stomach (St) but not in the intestine (I) and spleen (Sp) in mice at 21-day-old (right). b. Detection of the truncated Atbf1 mRNA by real-time RT-PCR in the brain, liver, heart, intestine, kidney and spleen but not in the lung and stomach of the Atbf1 heterozygous mice. c. Molecular and histological analyses of mouse kidneys from both Atbf1 wildtype (wt/wt) and heterozygous (wt/Δ7&8) mice. HE, representative images from Hematoxylin and Eosen stained sections. IHC staining confirmed reduced Atbf1 expression, increased Ki-67 expression and attenuated CK18 expression in the Atbf1 heterozygous mice (wt/Δ7&8) when compared to wildtype mice (wt/wt). All scale bars are 100 μm.