[CITATION][C] Hepatocyte‐specific expression of Cre recombinase

C Kellendonk, C Opherk, K Anlag, G Schütz… - genesis, 2000 - Wiley Online Library
C Kellendonk, C Opherk, K Anlag, G Schütz, F Tronche
genesis, 2000Wiley Online Library
To obtain hepatocyte-specific recombination of a DNA segment flanked with LoxP sites, we
generated transgenic mice that express the Cre-recombinase open reading frame (ORF)
under the control of both the mouse albumin regulatory elements and the-fetoprotein
enhancers (AlfpCre transgene). This configuration was chosen to mimic the genomic
organization of the mouse albumin gene. In mammals, the albumin gene is located
upstream of the-fetoprotein (AFP) gene, and its expression is thought to be influenced by the …
To obtain hepatocyte-specific recombination of a DNA segment flanked with LoxP sites, we generated transgenic mice that express the Cre-recombinase open reading frame (ORF) under the control of both the mouse albumin regulatory elements and the-fetoprotein enhancers (AlfpCre transgene). This configuration was chosen to mimic the genomic organization of the mouse albumin gene. In mammals, the albumin gene is located upstream of the-fetoprotein (AFP) gene, and its expression is thought to be influenced by the AFP enhancers located between both genes (Wen et al., 1991). We engineered a liver-specific expression vector pictured in Fig. 1. It contains several regulatory elements, the mouse albumin enhancer (10.5 to 8.5 kb), the albumin promoter (Pinkert et al., 1987) and the three mouse AFP enhancers (Hammer et al., 1987; Godbout et al., 1988). This liver-specific Cre expression vector differs from recently reported ones based on the use of only the albumin regulatory elements (Postic et al., 1999; Yakar et al., 1999). After injecting the construct into FVB/N oocytes, we obtained and analyzed four independent transgenic lines. All of them expressed Cre recombinase mRNA in liver as seen by immunohistochemistry, using a polyclonal Cre antibody (Kellendonk et al., 1999) and by Northern blot analysis (not shown). To determine the recombination pattern, the four transgenic lines were further crossed with mice carrying a modified allele of the glucocorticoid receptor (GR) in which the third exon has been flanked by two LoxP sites (GRLoxP allele; Tronche et al., 1999). This exon encodes the first zinc-finger of the GR DNA binding domain. Cre-mediated recombination between the LoxP sites will delete the exon and therefore inactivate the GR gene. Southern blot analysis of genomic DNA from different organs (Fig. 2A) of heterozygous GRLoxP animals carrying the AlfpCre transgene showed abundant recombination in the liver but no recombination in other organs. The Alfp proved a robust expression vector to drive hepatocyte-specific gene expression in transgenic mice since all four tested lines (AlfpCre 3, 4, 7, 8) displayed liver-specific recombination (Fig. 2A). This indicates that the chromosomal integration site is probably not affecting the expression pattern of the Alfp vector.
The liver is composed of several cell types including hepatocytes, which are the most abundant cells, biliary duct cells, endothelial cells, Kupffer cells, and Ito cells. To visualize on a cellular level the recombination pattern, we followed the disappearance of GR protein in AlfpCre mice, which are homozygous for the GRLoxP allele. For this study, only line AlfpCre7 was further analyzed. Using an antiserum directed against GR, we stained paraffin sections from the liver of both GRLoxP/LoxP-AlfpCre and GRLox/LoxP control animals by immunohistochemistry. GR protein expression could not be detected in hepatocytes and biliary duct cells. This is consistent with the fact that both cell types are probably derived from a common progenitor cell that expresses the albumin gene (Marceau et al., 1994). Based on the GR expression data, we believe that all hepatocytes carry recombined GR alleles. Nonepithelial cells such as endothelial and Kupffer cells showed normal GR expression (Fig. 2B). In mice, the albumin gene starts its expression during embryogenesis shortly after the appearance of the liver bud (day 9.5 pc) and weak mRNA levels increase following liver development (Cascio and Zaret, 1991). To detect the onset of recombination in Alfp-Cre GRLoxP/mice, we performed a PCR with genomic DNA that had been isolated from the head and the fetal liver (data not shown …
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