Generation of a conditionally null allele of hnf4α

The hepatocyte nuclear factor 4 (hnf4 ) is a member of the steroid hormone receptor family that plays an important role in regulation of hepatic gene expression (Li et al., 2000; Sladek et al., 1990). It has been shown that a complete loss of hnf4 results in embryonic lethality caused by defects in visceral endoderm function (Chen et al., 1994; Duncan et al., 1997). This early embryonic lethality can be overcome by complementing the hnf4 null embryos with an hnf4 / visceral endoderm by tetraploid aggregation (Duncan et al., 1997; Li et al., 2000). Unfortunately, we were unable to generate latestage embryos using this procedure that were suitable for the analysis of hnf4 ’s role in liver morphogenesis. Here we describe the generation of a conditionally null allele of hnf4 that will be suitable for this purpose. Figure 1a shows that we employed a two-step Cre/ loxP recombination strategy to delete exon 2 of the hnf4 gene that encodes a portion of the hnf4 DNA binding domain (Hadzopoulou et al., 1997). A targeting vector containing three loxP elements was constructed. The first loxP site was introduced into a SmaI restriction endonuclease cut site in intron 1 and another two loxP elements that flanked a thymidine kinase/neomycin phosphotransferase (tk/neo) expression cassette were introduced into an Asp718 site in intron 2 (Fig. 1a). The targeting vector also contained a diphtheria toxin (DT) gene that selected against random integration events. Following electroporation of the targeting construct into R1 ES cells, G418-resistant ES cell clones were tested for homologous recombination at the hnf4 locus by genomic Southern blot analysis (Fig. 1b) (Nagy et al., 1993). After digestion with EcoRV, a 700-bp probe outside the 5 arm of the targeting vector, probe A, detects an endogenous 10.2-kb fragment while a correctly targeted allele should contain an additional 6.8-kb fragment due to the introduction of a novel EcoRV site from tk/neo. Similarly, a 600-bp probe from outside the 3 end of the targeting vector, probe B, should detect the same wild-type 10.2-kb and a targeted 6.4-kb fragment. Twenty percent of G418-resistant clones collected were found to contain a correctly targeted allele, an example of which is shown in Figure 1b. Next, the tk/neo expression cassette was removed by transient expression of Cre recombinase, and successfully recombined clones were identified by selection for growth in gancyclovir, as described previously (Sund et al., 2000). As shown in Figure 1a, two possible outcomes could arise. Recombination between loxP elements b and c would generate an allele containing hnf4 exon 2 flanked by loxP elements. In contrast, recombination between sites a and c would replace exon 2 with a single loxP site. These two alleles are referred to as hnf4 loxP/ and hnf4 loxP / to indicate these events. As shown in Figures 1a and b, these recombination events can be distinguished by Southern blot analysis of genomic DNA that has been digested with EcoRV and probed with probe A. We have shown previously that loss of hnf4 function can be determined in ES cell embryoid bodies by measuring changes in hnf4 -mediated gene expression (Duncan et al., 1997). We, therefore, generated ES cell lines in which both copies of hnf4 exon2 had been deleted through recombination between flanking loxP elements by repeating the targeting procedure discussed above on the remaining wild-type allele. Figure 1b shows Southern blot analysis confirming the genotype of hnf4 loxP /loxP ES cell lines in which both alleles of hnf4 exon 2 are replaced by single loxP elements. This is the genotype expected of hepatocytes that have successfully undergone Cre-mediated recombination. Figure 2a shows RT-PCR analysis of steady state mRNA levels of genes known to require hnf4 for expression in ES cell embryoid bodies and in hepatocytes (Duncan et al., 1997; Li et al., 2000). As predicted, the homozygous replacement of hnf4 exon 2 with loxP elements prevented expression of apolipoproteins-B and -CII and aldolase B (Fig. 2a). This assay confirms that deletion of exon 2 results in loss of hnf4 function. Hnf4 loxP/ ES cells were aggregated with CD-1 morulae to generate chimeras (Wood et al., 1993). Chimeric males were then mated to CD-1 females to transmit the hnf4 loxP allele through the germ line. Hnf4 loxP/ mice were bred inter se and also mated to Albafp.Cre transgenic mice that express Cre recombinase in the developing fetal liver (K.H.K., unpublished). Hnf4 loxP/ ;Albafp.Cre offspring were then mated with hnf4 loxP/loxP mice to generate hnf4 loxP/loxP;Albafp.Cre and hnf4 loxP/ :AlbafpCre embryos. Paraffin sections from E15.5 livers isolated from these embryos were stained with anti-hnf4 antibodies by immunohistochemistry. Figure 2b shows that while strong