Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on May 11, 2005
Human Molecular Genetics 2005 14(13):1839-1850; doi:10.1093/hmg/ddi190

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A mouse model of tuberous sclerosis 1 showing background specific early post-natal mortality and metastatic renal cell carcinoma

Catherine Wilson¹, Shelley Idziaszczyk¹, Lee Parry¹, Carol Guy¹, David F.R. Griffiths², Edward Lazda², Rosemary A.L. Bayne^3,, Andrew J.H. Smith³, Julian R. Sampson¹ and Jeremy P. Cheadle^1,*

¹Department of Medical Genetics, Cardiff University, Heath Park, Cardiff CF14 4XN, UK, ²Department of Pathology, University Hospital of Wales, Heath Park, Cardiff CF14 4XN, UK and ³Gene Targeting Laboratory, Institute for Stem Cell Research, University of Edinburgh, The King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK

^* To whom correspondence should be addressed. Tel: +44 2920742652; Fax: +44 2920746551; Email: cheadlejp{at}cardiff.ac.uk

Received March 17, 2005; Accepted May 6, 2005

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either the TSC1 or the TSC2 genes and characterized by the development of benign hamartomatous growths in multiple organ systems. We have inactivated Tsc1 in the mouse germ line by gene targeting in ES cells and confirmed that the mutant allele (Tsc1^–) has a recessive embryonic lethal phenotype. We found that a significant number (27%) of heterozygous (Tsc1^+/–) mice on the C57BL/6 background died before weaning (P=0.014) and show that these mice die in the post-natal period (P=0.033), normally at 1–2 days, from unknown causes. Forty-four percent (7/16) of Tsc1^+/– mice on a C3H background developed macroscopically visible renal lesions as early as 3–6 months, increasing to 95% (37/39) by 15–18 months. Renal lesions progressed from cysts through cystadenomas to solid carcinomas. Eighty percent (16/20) of Tsc1^+/– mice on a Balb/c background exhibited solid renal cell carcinomas (RCC) by 15–18 months and in 41%, RCCs were 5 mm, resulting in grossly deformed kidneys. Some RCCs had a sarcomatoid morphology of spindle cells in whorled patterns and metastasized to the lungs. We detected loss of the wild-type Tsc1 allele and elevated levels of p-mTOR and p-S6 in lesions from Tsc1^+/– mice. This new murine model of hamartin deficiency exhibits a more severe phenotype than existing models.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the development of benign hamartomatous growths in multiple organs and tissues including the brain, heart, skin, lungs and kidneys (1). Approximately 60% of cases with TSC are sporadic, representing new mutations, and TSC occurs in at least one in 10 000 live births, without apparent ethnic clustering (2). Patients with TSC harbour a wide variety of germline mutations in either the TSC1 gene on chromosome 9q34 or the TSC2 gene on chromosome 16p13.3 (reviewed in 3). Several studies of TSC-associated renal and some extra-renal lesions demonstrate that hamartomas develop according to Knudson's ‘two-hit’ hypothesis with complete loss of either TSC1 or TSC2 (4). Hamartin, the TSC1 encoded protein, and tuberin, the TSC2 encoded protein, associate in vivo forming a tumour suppressor complex (5). Drosophila and mammalian studies have demonstrated that the hamartin–tuberin complex functions within the PI3K-Akt-mTOR (mammalian target of rapamycin) pathway and regulates nutrient and growth factor signalling to mTOR (reviewed in 6). Downstream, mTOR acts as a regulator for S6 kinase-1 (S6K1) and eIF-4E binding protein-1 (4E-BP1), which are responsible for cell growth and proliferation and become constitutively activated when the hamartin–tuberin complex is rendered dysfunctional.

Two groups have generated mouse lines with constitutively inactivated Tsc1 (7,8) and Tsc2 (9,10) alleles. Consistent with the phenotype severity differences that Jones et al. (11) and Dabora et al. (12) have seen in humans with mutations in TSC1 and TSC2, mice heterozygous for the inactivated Tsc1 allele (Tsc1^+/– mice) developed renal lesions by 15–18 months, whereas mice heterozygous for a mutant Tsc2 allele (Tsc2^+/– mice) developed such lesions by 6 months (7). In addition, a naturally occurring rat model of Tsc2 inactivation (the ‘Eker rat’) has been identified that is pre-disposed to renal adenoma and carcinoma (13). Eker rats also develop pituitary adenomas, uterine leiomyomas and leiomyosarcomas and splenic haemangiomas (14) and a variety of brain lesions at low frequency, including lesions resembling human TSC-associated subependymal nodules and cortical tubers (15).

Here, we describe the development and characterization of another mouse model for tuberous sclerosis-1, to help determine the mechanisms by which hamartin deficiency leads to TSC-associated disease. This new model is more severely affected than the published Tsc1 murine models, and heterozygotes exhibit increased early post-natal mortality and metastatic renal cell carcinoma (RCC).

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Generation of Tsc1^+/– mice
We previously cloned, sequenced and characterized the murine Tsc1 locus from the 129/Ola strain (16). We constructed a replacement-type targeting vector for homologous recombination in mouse embryonic stem (ES) cells. This vector was designed to inactivate an endogenous Tsc1 gene by deleting an internal region of the gene comprising the 3' half of exon 6 and all of exons 7 and 8 and substituting this with a ß-galactosidase reporter/neomycin selection [(TAG)₃/IRESlacz-polyA/loxP/MC1neo-polyA/loxP] cassette (17) (Fig. 1A). Insertion of this cassette was predicted to truncate the Tsc1 coding sequence in exon 6 by introducing TAG stop codons in all three frames and to create a bicistronic mRNA in which expression of the ß-galactosidase coding sequence was brought under the control of Tsc1 transcriptional regulatory sequences via an internal ribosome entry site (IRES). The linearized vector was electroporated into E14 Tg2aIV ES cells and correctly targeted clones identified by Southern blot analysis of EcoRI-digested DNA, using 5' and 3' probes external to, and flanking, the vector homology arms. Targeting efficiency was 3.4%. Chimeric male mice generated from two independently derived targeted ES cell lines (I/F5 and I/G11) gave offspring with the ES cell coat colour in test crosses with C57BL/6 females, and germ line transmission of the targeted allele was confirmed in 50% of these by Southern blot analysis on tail DNA (Fig. 1B and C). Further confirmation of successful targeting was obtained by carrying out long distance PCR using a primer within the neomycin coding sequence and a primer in exon 10 (outside the region of homologous recombination) and sequencing this 3' recombination product (Fig. 1D).

View larger version (57K): [in this window] [in a new window]   Figure 1. (A) Schematic illustration of the Tsc1 replacement targeting vector (upper panel), the wild-type Tsc1 locus (middle panel) and the targeted locus (lower panel). The vector comprised cloned 129/Ola genomic DNA consisting of a 1.3 kb region spanning exon 5 and part of exon 6 of Tsc1 for the 5' homology arm and a 1.9 kb region spanning exon 9 of Tsc1 for the 3' homology arm, flanking a (TAG)3/IRES-lacz-polyA/loxP/MC1neo-polyA/loxP reporter/positive selection cassette in the same transcriptional orientation. A negative selection cassette, MC1-tk2, consisting of two copies of the HSV thymidine kinase (tk) gene was positioned at the outer end of the 3' homology arm. The vector was linearized at a unique SalI site in the plasmid backbone. The regions of homology in which cross-overs (denoted X) can occur between the linearized vector and the Tsc1 locus to result in a replacement event are indicated by the dashed lines. The predicted targeted locus contains the reporter/positive selection cassette inserted into exon 6 of Tsc1 and introduces stop codons (TAG)3 into all three reading frames of the Tsc1 coding sequence. The targeting event also simultaneously deletes the 3' part of exon 6 and all of exons 7 and 8 of Tsc1. Exons are shown as numbered black rectangles, introns as a thick black line, flanking genomic regions as a thick dashed line and plasmid vector sequence as a thin black line. The reporter/selection cassette is shown as a large light grey striped rectangle (IRES-lacz component) and a large filled light grey rectangle (MC1neo-polyA component), with dark grey triangles indicating loxP sites. The negative selection cassette is shown as a grey chequered rectangle. The positions of relevant EcoRI sites and the predicted sizes of EcoRI restriction fragments (thin dashed lines with arrows) detected with the 5' and 3' probes (small black striped rectangles) before and after targeting and the long distance (LD) PCR products spanning the wild-type locus and the 3' recombination junction are shown below the wild-type and targeted loci. (B) Southern blot analysis of EcoRI digested DNA from wild-type (wt) and Tsc1+/– mice and Tsc1–/– embryos, analysed with the 5' probe. The 18.7 kb wild-type fragment is present in only wild-type and Tsc1+/– mice and the 16.7 kb targeted fragment is present in only Tsc1+/– mice and Tsc1–/– embryos, as expected. (C) Southern blot analysis of EcoRI digested DNA from wild-type (wt) and Tsc1+/– mice and Tsc1–/– embryos, analysed with the 3' probe. The 7.8 kb wild-type fragment is present in only wild-type and Tsc1+/– mice, whereas the 4.4 kb targeted fragment is present in only Tsc1+/– mice and Tsc1–/– embryos, as expected. (D) Multiplex PCR of long distance PCR products generated from the wild-type locus (using primers in exon 8 and intron 8 to amplify a 1.7 kb product) and from the targeted locus (using primers in the neomycin cassette and exon 10 to amplify a 2.3 kb product). The 1.7 kb wild-type fragment is present in only wild-type and Tsc1+/– mice, whereas the 2.3 kb targeted fragment is present in only Tsc1+/– mice and Tsc1–/– embryos.

 
We initially carried out a comprehensive molecular and phenotypic analysis of Tsc1^+/– mice generated from both of these chimeras; however, as the derivatives from the two ES cell clones did not exhibit any obvious differences, only one line (derived from the I/F5 cells) was analysed further.

Functional consequence of the targeting event
RT–PCR expression analysis was performed using primers predicted to amplify both the wild-type Tsc1 transcript (exons 4–7) and the mutant transcript (both exon 4 to IRES and internal to lacz) using RNA extracted from a range of tissues from Tsc1^+/– mice. Unexpectedly, products were only generated using the exon 4–7 primer set (specific to the wild-type transcript). Consistent with a failure to detect lacz mRNA expression from the mutant allele, we did not detect ß-galactosidase activity in kidneys from Tsc1^+/– mice, although we did detect ß-galactosidase activity in kidneys from Pkd1^+/– mice that also carried a lacz expression construct (18) (data not shown).

RT–PCR was then performed with primers in exons 4 and 10, which lie outside the regions of homologous recombination, so that we could detect any aberrant expression products from the modified locus. A product corresponding to correctly spliced exon 4–10 transcripts was detected in RNA extracted from wild-type and Tsc1^+/– mice but not in RNA extracted from Tsc1^–/– embryos. A strongly expressed truncated transcript was also detected in RNA extracted from Tsc1^+/– mice and was the only product detected in Tsc1^–/– embryos. This aberrant transcript was not present in RNA extracted from wild-type mice. Cloning and sequencing of these products confirmed the presence of normal, correctly spliced Tsc1 transcripts in wild-type and Tsc1^+/– mice and revealed an unpredicted spliced transcript that lacked exons 6–8, and joined exons 5 and 9, in Tsc1^+/– mice and Tsc1^–/– embryos (Fig. 2A and B). The aberrantly spliced transcript generated from the targeted allele causes a shift in the reading frame, leading to the introduction of a premature termination codon at the eighth codon of exon 9 of Tsc1 (Fig. 2C).

View larger version (30K): [in this window] [in a new window]   Figure 2. Functional consequence of homologous recombination with the knockout vector. (A) RT–PCR analysis using primers in exons 4 and 10 of Tsc1. Full length, normally spliced products of 900 bp were detected only in RNA extracted from wild-type and Tsc1+/– mice. Truncated products were detected only in RNA extracted from Tsc1+/– mice and Tsc1–/– embryos. Further faint bands that were not seen in wild-type mice and Tsc1–/– embryos were detected in RNA from Tsc1+/– mice; these were shown to correspond to heteroduplex species formed between wild-type and mutant RT–PCR products. (B) Sequence analysis of cloned RT–PCR products from Tsc1+/– mice and Tsc1–/– embryos identified the presence of a mutant Tsc1 transcript lacking exons 6–8 and fusing exons 5 and 9 as a consequence of an aberrant splicing event, thus excluding the coding sequence of the 5' region of exon 6 and the IRES-lacz sequence in the mRNA. DNA sequences are indicated as in Figure 1 and RNA exon sequences are indicated by grey rectangles. (C) Mutant transcripts were predicted to cause a shift in the reading frame and introduce a premature termination codon at the eighth codon in exon 9 of Tsc1 and were thus considered to be inactivating.

 
Tsc1^–/– mice die during embryonic development
Tsc1^+/– intercrosses (Tsc1^+/–xTsc1^+/–) were set up using F1 mice generated from the chimera test crosses (50% 129ola/50% C57BL/6). From 11 intercrosses, 138 progeny were obtained, 47 of which were Tsc1^+/+, 86 were Tsc1^+/– and none were Tsc1^–/– (five mice failed to be genotyped). This ratio differs significantly from expected (P<0.0001), indicating that the Tsc1 mutant allele has a recessive, embryonic lethal phenotype. Tsc1^+/– mice were backcrossed (Tsc1^+/–xTsc1^+/+) onto a C57BL/6 background (n4), and 15 Tsc1^+/– intercrosses were set up for timed matings to study the embryonic development of Tsc1^–/– animals. Most Tsc1^–/– embryos died between embryonic day (E) 10.5 and E12.5, and no viable null embryos were observed at E13.5 (Table 1). Tsc1^–/– embryos were generally smaller and developmentally retarded when compared with wild-type and heterozygous littermates (e.g. mean size of Tsc1^–/–, Tsc1^+/+ and Tsc1^+/– embryos was 2.8, 4.3 and 3.8 mm at E10.5, respectively). Two out of 12 (17%) null embryos displayed exencephaly and, at E12.5, two out of 2 null embryos had abnormal vacuolation of myocardial cells. No liver hyperplasia was observed.

View this table: [in this window] [in a new window]   Table 1. Genotypes of embryos obtained from Tsc1+/– intercrosses

 
Some Tsc1^+/– mice on a C57BL/6 background die post-natally
Tsc1^+/– mice were backcrossed (n3) with inbred (wild-type) C57BL/6 mice. Genotyping showed that out of the 253 offspring that survived until weaning, 103 were Tsc1^+/– and 142 were wild-type (eight failed to be genotyped), which is significantly different from the expected 1:1 ratio (P=0.014) (Table 2). This indicated 27% excess mortality among Tsc1^+/– mice before weaning. To investigate the point at which these Tsc1^+/– mice died (in utero or post-natally), additional crosses between Tsc1^+/– mice at this backcross generation and wild-type C57BL/6 mice were set up and offspring genotyped at birth. Out of 184 new-born pups, 95 were Tsc1^+/– and 88 were wild-type (one mouse failed to be genotyped), thus excluding in utero mortality. Close examination of another cohort of mice revealed that a significant proportion of animals died soon after birth. We collected and genotyped the carcasses of 37 of these animals and showed that 25 were Tsc1^+/– and 12 were wild-type, which is significantly different from expected (P=0.033) (Table 2). Twenty-two (88%) of the Tsc1^+/– pups died at 1–2 days after birth and the remaining three died at 2 weeks after birth. Histological analysis of the heart, brain, kidneys, liver, digestive tract, thymus and pancreas from 10 of these animals did not reveal any morphological differences when compared with age-matched wild-type littermates.

View this table: [in this window] [in a new window]   Table 2. Genotypes of offspring from Tsc1+/– and Tsc1+/+ crosses (n3 C57BL/6)

 
We backcrossed (n3) Tsc1^+/– mice onto two further genetic backgrounds (C3H and Balb/c). No evidence of increased post-natal mortality of Tsc1^+/– mice was observed on either of these backgrounds (144 Tsc1^+/– and 138 Tsc1^+/+ mice on the Balb/c background and 120 Tsc1^+/– and 120 Tsc1^+/+ mice on the C3H background survived until weaning), indicating that this phenotype was specific to the C57BL/6 background.

Renal pathology
We examined the kidneys of mice at 3–6, 9–12 and 15–18 months by macroscopic inspection and microscopic analysis of five sections 200 µm apart. On a C3H background, 44% (7/16) of Tsc1^+/– mice developed macroscopically visible renal lesions by 3–6 months, increasing to 95% (37/39) by 15–18 months (Table 3). On the Balb/c and C57BL/6 backgrounds, the phenotype was less dramatic with, respectively, 13% (2/16) and 8% (1/13) of Tsc1^+/– mice developing macroscopically visible renal lesions by 3–6 months and 88% (35/40) and 67% (29/43) by 15–18 months (Table 3). Microscopically, all (10/10) Tsc1^+/– mice on a C3H background had renal lesions by 3–6 months (average of 10.2 lesions per mouse) compared to 90 (9/10) (6.2 lesions) and 60% (6/10) (2.0 lesions) on Balb/c and C57BL/6 backgrounds, respectively. By 15–18 months, all mice had microscopically visible lesions regardless of background (with 29, 26.8 and 14.8 lesions per mouse on C3H, Balb/c and C57BL/6 backgrounds, respectively). None of the wild-type littermates had macro- or microscopic kidney lesions by 15–18 months.

View this table: [in this window] [in a new window]   Table 3. Frequency and size of macroscopic renal lesions (cystic and solid) in Tsc1+/– mice grouped according to background and age

 
Renal lesions varied from pure cysts through to solid anaplastic carcinomas and were classified as cysts (solitary cysts lined with one layer of epithelium), atypical cysts (cysts with single papillary projections into the lumen), branching cysts (cysts with branching papillary projections), mixed cystic/solid carcinomas or solid carcinomas (examples shown in Fig. 3B and C). The distribution in the size of the lesions, with simple cysts tending to be smaller and more numerous than branching cysts, mixed cysts and solid carcinomas (Table 4), suggests that some small cysts progressed to carcinomas. In addition, there is a preponderance of cysts in younger animals and a morphological continuity of lesions further supporting this progression. A majority of lesions expressed gelsolin, a proposed marker for Tsc-associated renal lesions (9), with staining intensities similar to distal tubules and collecting ducts; however, some did not (Fig. 3D). By 15–18 months, Tsc1^+/– mice on a C3H background had significantly more cystic lesions (simple, atypical and branching cysts) when compared with mice on a C57BL/6 background (P=0.013) and those on a Balb/c background had significantly more solid lesions (mixed cystic/solid lesions and solid carcinomas) when compared with mice on C3H (P=0.014) or C57BL/6 (P=0.004) backgrounds.

View larger version (114K): [in this window] [in a new window]   Figure 3. Renal cystadenomas in Tsc1+/– mice on a C3H background. (A) Kidneys from an 18-month-old mouse showing macroscopic renal cystadenomas (indicated by an arrow). (B) Microscopic view of a cyst. (C) Microscopic view of a complex cystadenoma with branching papillae and an adjacent carcinoma. (D) Immunostaining with an anti-gelsolin antibody showed gelsolin expression (indicated by brown staining) in a papillary cystadenoma (arrow on left) and lack of expression in a solid carcinoma (arrow on right). Macroscopic bars are 1 cm and microscopic bars are 200 µm.

 

View this table: [in this window] [in a new window]   Table 4. Number and histological classification of microscopic renal lesions in Tsc1+/– mice grouped according to background and size

 
Tsc1^+/– mice develop RCCs, resulting in grossly deformed kidneys
Eighty percent (16/20) of Tsc1^+/– mice on a Balb/c background exhibited solid RCCs by 15–18 months, and in 41% (17/40), RCCs were 5 mm, which resulted in grossly deformed kidneys of up to 2.5 cm in size (Fig. 4A–C). One mouse displayed this distinctive phenotype as early as 11 months. Tsc1^+/– mice on C57BL/6 and C3H backgrounds rarely harboured RCCs 5 mm at 15–18 months (7 and 3%, respectively, P<0.002). Four out of nine (44%) RCCs 5 mm (analysed from different mice) had a sarcomatoid morphology consisting of spindle cells with nuclear anaplasia arranged in whorled patterns (Fig. 4D); three of these had metastasized to the lungs (the only lung tumours observed in this study) (Fig. 4E and F).

View larger version (143K): [in this window] [in a new window]   Figure 4. RCC in Tsc1+/– mice on a Balb/c background resulting in grossly deformed kidneys and metastases in the lungs. (A and B) Paired kidneys from 18-month-old Tsc1+/– mice with RCC 5 mm. (C) Section through a kidney displaying RCC. (D) Microscopic view of a sarcomatoid RCC with elongated sheets of spindle cells intermingled with more typical carcinomatous areas. (E) Macroscopic and (F) microscopic RCC metastases in the lungs (the metastatic nature of lesions was determined by the histological appearance of cells with renal tubular morphology in the lungs). Macroscopic bars are 1 cm and microscopic bars are 200 µm.

 
Extra-renal tumours
Microscopic liver haemangiomas were observed in 18% of 15–18 month Tsc1^+/– mice, regardless of background or sex; these lesions were not seen in wild-type littermates. Histologically, haemangiomas consisted of abnormal vascular channels and a proliferation of smooth muscle cells. Liver hepatomas were seen at equal frequency (24%) in Tsc1^+/– and wild-type littermates, suggesting that their pathogenesis is independent of Tsc1 mutation status. One Tsc1^+/– mouse (Balb/c background) had a splenic haemangioma and two (C3H background) had isolated uterine leiomyoma/leiomyosarcomas.

Molecular and immunological analysis of lesions
DNA was extracted from laser capture microdissected kidney, liver, uterine and lung (a kidney metastasis) lesions and analysed for loss of heterozygosity (LOH) at the Tsc1 locus using an IRES (mutant specific) and exon 8 (wild-type-specific) quantitative PCR assay. Loss of the wild-type allele was observed in five out of 12 renal lesions, two out of five hepatic haemangiomas, one out of two uterine lesions and one out of one lung lesion (a kidney metastasis) (Fig. 5A). We investigated the expression levels of mTOR, phosphorylated (p)-mTOR and p-S6 ribosomal protein (p-S6) in the renal lesions. Western blot analysis showed that although levels of mTOR were the same in normal kidney and adjacent solid tumours, levels of p-mTOR and p-S6 were increased substantially in all lesions examined (Fig. 5B). Immunohistochemical analysis also showed increased expression of p-mTOR and p-S6 in renal cystadenomas when compared with adjacent normal tissue (Fig. 5C and D).

View larger version (68K): [in this window] [in a new window]   Figure 5. Molecular and immunological analysis of renal lesions in Tsc1+/– mice on a Balb/c background. (A) Detection of LOH in a microdissected renal tumour. Loss of the wild-type Tsc1 allele in the tumour, but not in adjacent normal tissue, is indicated by an arrow (WT, wild-type allele; Mut, mutant allele). The red trace is an ABI GS500 internal size standard. (B) Western blot analysis showing relative expression levels of mTOR, phosphorylated mTOR (P-mTOR) and phosphorylated S6 ribosomal protein (P-S6) in tumour (a cystadenoma) (T) and normal (N) kidney tissue. (C and D) Immunostaining of a papillary cystadenoma using (C) an anti-phospho-mTOR antibody and (D) an anti-phospho-S6 ribosomal protein antibody. Expression of phospho-mTOR and phospho-S6 around the periphery of the cyst is indicated by brown staining. Microscopic bars are 200 µm.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
We have generated mice bearing a targeted disruption of Tsc1 by deleting part of exon 6 through to exon 8, with the concurrent insertion of a reporter/selection cassette. This targeted modification should have expressed a bicistronic RNA transcript containing Tsc1 and IRES-lacz sequences, which was terminated at the polyadenylation site in the cassette, with the Tsc1 open reading frame truncated by a stop codon introduced at the beginning of the cassette sequence. However, we found that Tsc1^–/– embryos only expressed Tsc1 transcripts generated as a consequence of an aberrant splicing event fusing exons 5 and 9; therefore, excluding exon 6 and the IRES-lacz sequence. The exon 5–9 fusion alters the Tsc1 reading frame, which is then predicted to prematurely terminate at a TGA codon in exon 9; therefore, any protein produced from this allele would lack all of the known functional and interacting domains of hamartin (reviewed in 3). Our results suggest that in the targeted allele, the exon 9 splice acceptor is utilized in preference to that of exon 6 and, therefore, a transcript containing the IRES-lacz sequence is not produced efficiently. This explains our failure to detect ß-galactosidase activity in kidneys from Tsc1^+/– mice.

We have confirmed that our targeted mouse strain is a valid model for tuberous sclerosis-1 deficiency by several criteria: (i) Tsc1^–/– embryos die in utero at a similar stage to other murine models of Tsc1-deficiency (7,8); (ii) somatic mutation analysis of DNA extracted from microdissected renal and extra-renal lesions showed loss of the wild-type Tsc1 allele and (iii) western blot analysis and immunostaining showed increased levels of phosphorylated mTOR and phosphorylated S6 ribosomal protein in kidney lesions [in agreement with the findings of Kwiatkowski et al. (8)].

We found that 27% of Tsc1^+/– mice on a C57BL/6 background died before weaning, and this mortality appeared to occur in the post-natal period, primarily 1–2 days after birth. Although it is unclear why these animals die, it is interesting to note that Stoyanova et al. (19) reported substantial differences in the gene expression profiles of normal renal epithelial cells from TSC mutation carriers when compared with controls. Therefore, it is possible that heterozygosity for a single mutant allele may have effects on key metabolic processes in the context of specific genetic backgrounds, causing some mice to die without tumour development.

The Tsc1^+/– mice described in this study have a more severe renal phenotype when compared with the published models (7,8). By 3–6 months, 44% of our Tsc1^+/– mice on a C3H background developed macroscopically visible renal lesions and 100% had microscopic lesions. In contrast, Kobayashi et al. (7) failed to identify any macroscopic renal lesions in their Tsc1^+/– mice by 9–12 months. Furthermore, 80% of our 15–18 month mice on a Balb/c background showed progression to RCC, which is considerably higher than that described by Kwiatkowski et al. (8) (up to 31% depending on the background), and in 41%, RCCs were 5 mm, which resulted in grossly deformed kidneys. Some of these carcinomas had a sarcomatoid morphology of spindle cells in whorled patterns and, unlike other Tsc1^+/– mice, metastasized to the lungs.

Further molecular and cellular studies are necessary to determine whether the phenotypic differences between the Tsc1^+/– strain described herein and the published strains are due to the precise nature of the targeted modifications and/or to the presence or absence of modifiers in the various genetic backgrounds in which the mutated gene was studied. It should be noted that the Tsc1^+/– mice we have analysed were extensively backcrossed, so that any random genetic changes introduced during ES cell culture are likely to have been segregated from the targeted allele (20). In contrast, the Tsc1^+/– mice studied by Kobayashi et al. (7) and Kwiatkowski et al. (8) were F1s of mixed genetic backgrounds.

The phenotype of all reported Tsc1 knockout mouse models differs from humans with TSC, in whom benign angiomyolipomas are the most common renal lesion. Although the occurrence of RCC in humans with TSC is unusual, an association is recognized (21). The carcinomas are typically discovered at a young age and are thought to evolve from the lining of hyperplastic cysts. The proportion of sarcomatoid features in TSC-associated renal carcinomas (three out of six) is far greater than in sporadic RCC (1–2%) (1,22), and these renal carcinomas tend to behave aggressively. Our Tsc1^+/– mice may, therefore, prove an invaluable model to study this facet of the human disease.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Targeted inactivation of the Tsc1 gene and generation of Tsc1^+/– mice
A replacement-type targeting vector was constructed in pUC19 using 129/Ola cloned genomic DNA containing the Tsc1 gene (16). This comprised a 1.3 kb region spanning exon 5 and part of exon 6 of Tsc1 for the 5' homology arm and a 1.9 kb region spanning exon 9 for the 3' homology arm. A reporter/positive selection cassette, (TAG)₃/IRES-lacz-polyA/loxP/MC1neo-polyA/loxP, was inserted between the homology arms. This cassette (a newer version of that previously described (17), modified to contain loxP sites), therefore, disrupted exon 6, inserting TAG codons in all three reading frames at the junction and introduced a lacz reporter preceded by an IRES and followed by an SV40 polyadenylation sequence. This allows expression of the reporter from the IRES but under control of the endogenous targeted gene's promoter. The positive selection marker (MC1neo-polyA) contained in the cassette is independently expressed using its own enhancer/promoter and polyadenylation sequence. A negative selection cassette (MC1tk²), which consists of two copies of the HSV thymidine kinase gene, independently expressed using its own enhancer/promoter and polyadenylation sequence (23), was appended to the end of the right homology arm. The vector was linearized at a unique SalI site.

Seventy-five micrograms of linearized targeting construct was electroporated into 4.5x10⁷ E14 Tg2aIV ES cells (from 129/Ola strain). Cells were selected on 160 µg/ml G418 containing medium from 1 day after electroporation and then on G418 and Ganciclovir (GANC, 2.5 µM) containing medium after a further 4 days, to enrich for targeted clones by positive–negative selection (24). After 10 days, 700 G418 and GANC resistant colonies were picked into 96 well plates and subsequently replica plated for freezing and DNA isolation. DNA was digested with EcoRI and analysed by Southern blotting and hybridization with external 5' and 3' flanking probes to identify correctly targeted clones.

Tsc1^+/– ES cell clones were injected into C57BL/6 blastocysts and transferred into pseudo-pregnant (CBAxC57BL/6) females and chimeric pups identified. Male chimeras were mated with inbred C57BL/6 females to generate F1 mice (50% 129ola/50% C57BL/6) carrying the targeted mutation, identified by coat colour and subsequent genotyping on tail biopsied DNA. Tsc1^+/– F1 mice were backcrossed (n3) with inbred C57BL/6J Ola Hsd (C57BL/6), Balb/c Ola Hsd (Balb/c) and C3H HeN Hsd (C3H) mice (Harlan UK Limited).

Southern blotting and PCR genotyping
DNA was extracted from ES cells, yolk sacs, whole embryo paraffin sections or tail tips using QIAamp DNA mini kits (Qiagen). Southern blot analysis was performed on EcoRI digested DNA, separated on 0.7% agarose gels, blotted onto Zeta probe membrane (Biorad) and followed by hybridization at 65°C in modified Church and Gilbert buffer. A 0.7 kb PstI fragment cut from the vector pMC1neo-polyA, a 0.8 kb fragment containing exon 4 of Tsc1 and a 0.6 kb fragment containing exons 11 and 12 of Tsc1 were used as the neomycin probe, and 5' and 3' external flanking probes, respectively.

PCR genotyping of DNA from tail tips and embryo yolk sacs was performed by amplification of the wild-type and mutant Tsc1 alleles using the following primers in a 35 cycle PCR reaction with AmpliTaq gold DNA polymerase (Applied Biosystems): wild-type allele, 44F 5'-ATTCACCCGGAATTAGTGACTG-3' and 45R 5'-GCGTCCTCTTCTCCTTTTACAC-3' (1730 bp product) and for the mutant allele, either 16F 5'-AGCGTTGGCTACCCGTGATATTG-3' and 21R 5'-GTGTTGATGGGGAACTCAAACTCT-3' (2306 bp product) or sh2F 5'-GCCGAATATCATGGTGGAAA-3' and sh2R 5'-ACGAAGGTGATCAGGGAATG-3' (1100 bp product). Products were analysed on 1% agarose gels.

An independent PCR of DNA extracted from paraffin embedded embryo sections was performed to validate the yolk sac genotypes. PCR was performed by amplification of the wild-type and mutant alleles using the following primers in a 35 cycle PCR reaction: wild-type allele, EXON 8F 5'-TGCCTGGAAGCCCAGGAAGGT-3' and EXON 8R 5'-CTGCAGGGCCCATGGTGGTT-3' (183 bp product) and mutant allele, IRES F 5'-TAACGTTACTGGCCGAAG-3' and IRES R 5'-GTCGCTACAGACGTTGTT-3' (237 bp product).

RT–PCR analysis
RNA was extracted from tissues and embryos after snap freezing and grinding in liquid nitrogen using the RNAeasy kit (Qiagen). An aliquot of 50 ng–1 µg RNA was used for first strand cDNA synthesis using oligo(dT)₁₅ and Superscript II RNase H^– Transcriptase (Invitrogen Life Technologies). Second strand synthesis was carried out in 50 µl reaction volumes using 1 µl cDNA, 25 pmol primers, 0.2 mM dNTPs, 5 µl reaction buffer and 2U AmpliTaq Gold DNA polymerase in 35 cycle PCR reactions. Four reactions were carried out: (i) KO34 (exon 4 F) 5'-ACGTGGGCCTATGCTTGTCAACA-3' and KO35 (exon 7 R) 5'-AGCGCAGGAAGGAGACGAAGTTA-3', (ii) KO34 and IRES_R2 5'-TCCTTCAGCCCCTTGTTGAATAC-3', (iii) LACZ_F 5'-AAACCCTGGCGTTACCCAACTTA-3' and LACZ_R 5'-ACCGTCGATATTCAGCCATGTTC-3' and (iv) KO34 and KO21 (exon 10 R) 5'-GTGTTGATGGGGAACTCAAACTCT-3'. Products were analysed on 1% agarose gels, cloned into pGEM-T (Promega) and sequenced.

Animal care, necropsy and pathology
All procedures with animals were carried out in accordance with Home Office guidelines. Mice were tagged using microchips and tail tips were cut from mice for genotyping using a local anaesthetic. Mice were killed and analysed at three time points: 3–6, 9–12 and 15–18 months. Necropsy analysis included macroscopic examination of the brain, heart, lungs, kidneys, liver, spleen and uterus in all animals. Half of each organ was fixed and processed into paraffin wax, sectioned at 4 µm and stained with H&E for microscopic analysis (tissues from 10, 15 and 20 Tsc1^+/– mice and wild-type littermates on each background were analysed at 3–6, 9–12 and 15–18 months, respectively). The other half of the organ was snap frozen in liquid nitrogen-cooled isopentane for laser capture microdissection. To estimate the average number of microscopically visible kidney lesions per mouse, 10 Tsc1^+/– mice were analysed per background at 3–6, 9–12 and 15–18 months—five representative sections 200 µm apart from each half of kidney were stained with H&E and anti-gelsolin and inspected on an Olympus BX51 BF light microscope (lesions crossing more than one section were counted once and total numbers were doubled to generate a mean number per mouse). Animals that died before weaning were collected, genotyped and processed into paraffin wax, sectioned at 4 µm and stained with H&E for microscopic analysis.

Embryos were removed at 9.5, 10.5, 11.5, 12.5 and 13.5 days post-fertilization (noon on the day on which the vaginal plug was formed was defined as embryonic day 0.5). Yolk sacs were removed for genotyping and whole embryos were processed into paraffin wax, sectioned at 4 µm and stained with H&E for microscopic analysis.

Staining for ß-galactosidase activity
Staining was carried out on snap frozen tissue sections (10 µm) that were air dried for 30 min. Slides were washed in water and sections were fixed for 10 min in 0.5% gluteraldehyde. Sections were stained overnight at 20°C using the LacZ Reporter Assay Kit (Invitrogen), washed in PBS/MgCl₂ solution, counterstained in eosin, dehydrated and mounted with DPX.

Immunohistochemistry and immunoblotting
Paraffin sections were deparaffinized and rehydrated. For antigen retrieval, sections were boiled in 10 mM citrate buffer (pH 6.0) for 10 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 30 min. Immunostaining of paraffin sections was performed as described by the manufacturer using the rabbit VECTASTAIN ELITE ABC horseradish peroxidase kit (Vector Laboratories). Primary antibodies were incubated for 30 min at room temperature for anti-murine gelsolin (9) and overnight at 4°C for anti-phospho-S6 ribosomal protein (Ser240/244) and anti-phospho-mTOR (Ser 2448) (Cell Signalling Technologies). Sections were developed using DAB, counterstained in Gills haematoxylin, dehydrated and mounted with DPX.

Protein extraction from snap frozen tissues was performed using RIPA lysis buffer (Santa Cruz Biotechnologies) and homogenized on the fast prep system (Thermo Savant). Proteins were quantified using the Bradford assay (Bio-Rad). Fifty micrograms of total protein was analysed on 5 or 10% SDS–PAGE gels, blotted onto Immobilon-P PVDF membrane (Millipore) and incubated overnight with the primary antibodies, anti-mTOR, anti-phospho-mTOR (Ser 2448) or anti-phospho-S6 ribosomal protein (Ser 240/244) (Cell Signalling Technologies). Detection by enhanced chemiluminescence was performed after incubation with an anti-rabbit HRP secondary antibody (Amersham) for 1 h. Equal loading of protein was confirmed by incubation of membranes with an anti-ß actin antibody (Abcam Ltd) and Ponceau staining.

Somatic LOH analysis
Snap frozen tissue was sectioned at 10 µm onto PEN (PALM) membrane covered slides and stained with toluidine blue. Tumour and normal tissue were microdissected (PALM LCM) and DNA extracted using the QIAamp DNA micro kits (Qiagen). PCR of microdissected DNA was performed by simultaneous amplification of both the wild-type (EXON 8F-5'FAM labelled and EXON 8R) and the mutant (IRES F-5'FAM labelled and IRES R) alleles in a 25 cycle PCR reaction. An aliquot of 2 µl of PCR products was mixed with an ABI GS500 internal size standard and formamide loading buffer and run on an ABI3100 genetic analyser. Results were analysed using Genescan v.3.7 software. DNA extracted from eight different normal tissue sections from Tsc1^+/– mice was used to normalize the assay (comparison of wild-type: mutant allele peak heights). LOH was defined as a change in wild-type/mutant peak ratios 2.0.

Statistical analysis
Comparisons of numbers of Tsc1^+/– to Tsc1^+/+ mice were calculated using the ² test with one degree of freedom. Lesion counts per mouse were compared using the Kruskal–Wallis and Mann–Whitney confidence interval tests.

	ACKNOWLEDGEMENTS

 
We would like to thank L. Dobbie, J. Maynard, R. Snell, A. Hodges, N. Fleming, S. Hunter, J. Neal, A. Clarke, K. Davies, J. Dunstan, R. Davies, S. Macdonald, D. Adams, V. Moskvina, R. Newcombe and F. Dunstan for support or advice, D. Kwiatkowski for the anti-gelsolin antibody and R. Sandford for the Pkd1^+/– mice. This work was supported by the Tenovus, the Tuberous Sclerosis Alliance and the Wales Gene Park. The ISCR Gene Targeting Laboratory was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC).

Conflict of Interest statement. None declared.

	FOOTNOTES

 
Present address: MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK.

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