Several mouse models have been generated which are described below:
Homozygous deletion of FLCN in the mouse has been shown to be embryonic lethal (Baba et al., 2008; Chen et al., 2008). Therefore, two kidney-specific knockout mouse models were developed. The first contained a conditional FLCN allele and a cadherin 16 (KSP)-Cre transgene, which led to the deletion of exon 7 and the targeted inactivation of FLCN in the kidney (Baba et al., 2008). The second was also created using a Cre-lox system, which inactivated FLCN specifically in the kidney by deleting exons 3 and 4 (Chen et al., 2008).
In both studies, conditional homozygous inactivation of FLCN in the mouse kidney triggered the development of highly enlarged polycystic kidneys, which led to renal failure and death around three weeks after birth. These kidneys also exhibited increased phosphorylation of the indicator of mTOR activation, p-S6R, suggesting that a deficiency in FLCN leads to the activation of mTOR signalling in vivo (Baba et al., 2008; Chen et al., 2008).
Another mouse model with targeted inactivation of FLCN was generated by Hartman et al. (2009). These mice were created using a gene trap vector technique, where a β-galactosidase/neomycin (β-geo) cassette was integrated between exons 8 and 9 of the FLCN gene, which resulted in a truncated form of the Folliculin protein (Hartman et al., 2009). As expected, no homozygous FLCN mutant mice were identified. Furthermore, the heterozygous FLCN mutant mice produced developed renal cysts and neoplasia at 3-6 months, similar to those found in BHD patients (Hartman et al., 2009). Reduced p-S6R immunostaining was observed in the tumours of these mice, suggesting that mTOR activity within this system was in fact suppressed.
Subsequently, it was demonstrated that FLCN homozygous null mice die from embryonic day (E) 5.5 to E6.5, with multiple defects evident in their cellular structure (Hasumi et al., 2009). Hasumi et al. (2009) also showed that heterozygous FLCN knockout mice (generated by a Cre-lox deletion of exon 7) spontaneously develop cysts and solid tumours in their kidneys after 10 months of age, which are also similar to those seen in BHD patients. Conversely, these investigators noted an activation of mTOR signalling in the kidney tumours of these mice (Hasumi et al., 2009).
Further studies on a mouse model (with the in-frame β-geo insertion between exons 8 and 9 of FLCN) showed that homozygous deletion of FLCN caused embryonic lethality before E8.5 (Hudon et al., 2010). Heterozygous FLCN knockout mice from this model also developed renal cysts and tumours upon FLCN loss (Hudon et al., 2010). However, the authors noted that this loss of FLCN expression resulted in both elevated and reduced levels of p-S6R, depending on the cellular context- which may go some way in explaining the differing results observed in the earlier mouse models (Baba et al., 2008; Chen et al., 2008; Hartman et al., 2009; Hasumi et al., 2009; Hudon et al., 2010).
