Mouse models

Mouse models of both FLCN and FNIP1 have been generated and are described below:

FLCN mouse models

Baba et al., (2008) used gene targeting to generate a conditional Flcn, with loxP sites flanking exon 7. Deletion of exon 7 using Cre-recombinase produces a frameshift and generates a premature stop codon in exon 8. Homozygous deletion of Flcn in kidneys using a kidney-specific Cre-driver allele caused these mice to develop polycystic kidneys and to subsequently die of renal failure at 3 weeks post-partum.

Chen et al. (2008) also used gene targeting to generate a conditional Flcn allele, with loxP sites flanking exons 3 – 4 of the gene. This deletion removes the start codon, and is thus predicted to completely prevent the expression of Flcn. The authors demonstrated that constitutive homozygous deletion of Flcn resulted in embryonic lethality at E3.5-E8.5. The authors also found that homozygous deletion of Flcn specifically in kidneys caused mice to develop enlarged, polycystic kidneys and to subsequently die of renal failure at 3 weeks post-partum. Renal cyst cells cultured in vitro were able to form tumours when implanted into nude mice (Wu et al., 2015)

Hartman et al. (2009) generated a Flcn-null allele using gene trap technology: a β-galactosidase/neomycin (β-geo) cassette was integrated between exons 8 and 9 of the FLCN gene, resulting in a truncated form of the Flcn protein. No homozygous Flcn mutant mice were identified and heterozygous Flcn mutant mice developed renal cysts and neoplasia at 3-6 months, similar to those found in BHD patients.

Hasumi et al., (2009)  crossed mice carrying their previously developed FlcnFlox allele (Baba et al., 2008) with those carrying a β-actin-Cre construct to produce mice with ubiquitous Flcn knockout. Homozygous deletion of Flcn caused embryonic lethality before E9.5. Heterozygous Flcn knockout mice developed renal cysts and tumours of varied histology from around 12 months. The tumours showed vastly reduced Flcn expression, but increased activation of the PI3K-AKT-mTOR pathway.

Hudon et al. (2010) also generated a Flcn-null allele with a β-geo cassette insertion between exons 8 and 9 of Flcn, and showed that homozygous deletion of Flcn caused embryonic lethality before E8.5. Heterozygous Flcn knockout mice developed renal cysts and tumours, which were found to not express Flcn, suggesting that the wild-type allele had been inactivated in these tumours.

Chen et al., (2015) crossed mice carrying their previous developed FlcnFlox allele (Chen et al. 2008) with those carrying a Sglt2-Cre construct to produce mice with homozygous knockout of Flcn only in the kidney proximal tubules. These mice develop renal cysts and hyperplasia in the first 6 months, and renal tumours from around 6 months. The authors reported the development of multiple and often mixed histologies. Heterozygous mice rarely develop cysts or tumours indicating a low tumourigenicity.

FNIP1 mouse models

Fnip1-null mice have been generated by N-ethyl-N-nitrosourea (ENU) mutagenesis, which caused a 32bp deletion in exon 9 of Fnip1. These mice are viable and fertile, but display a block in B cell development (Park et al., 2012).

Two targeted Fnip1-knockout mouse models were also developed by Baba et al. (2012). One carries loxP-sites flanking exon 6, and the other carries a β-geo cassette insertion in intron 2. Since there were no phenotypic differences between the two models, they were used interchangeably during this study (Baba et al., 2012). These Fnip1 knockouts were both viable and fertile, however, they too displayed a block in B cell development.