Generation of a Conditional Disruption of the Tsc2 Gene

Transcription

1 ' 2007 Wiley-Liss, Inc. genesis 45: (2007) TECHNOLOGY REPORT Generation of a Conditional Disruption of the Tsc2 Gene Omar Hernandez, Sharon Way, James McKenna III, and Michael J. Gambello* Department of Pediatrics, Division of Medical Genetics, University of Texas Health Science Center, Houston, Texas Received 13 September 2006; Revised 11 December 2006; Accepted 17 December 2006 Summary: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 or TSC2 gene. Patients afflicted with TSC develop tumors in various organ systems, but cerebral pathology is particularly severe. Conventional gene disruption of the Tsc1 or Tsc2 gene in mice cause limited central nervous system pathology. Homozygous deletion of either gene causes midgestation lethality. To circumvent the homozygous lethality of the conventional Tsc2 knockout we have generated a conditional allele of the Tsc2 gene by homologous recombination in mouse ES cells. The homozygous Tsc2 flox/flox mice are identical to wildtype in many organs typically affected by TSC, especially the brain. Using this Tsc2 flox allele we have generated a null allele using Cre recombination. This allele will be useful in investigating TSC pathology with appropriate cell and organ specific Cre-transgenic mice. genesis 45: , VC 2007 Wiley-Liss, Inc. Key words: Tsc2; tuberin; Cre-loxP; tuberous sclerosis; conditional allele Tuberous Sclerosis Complex (TSC) is an autosomal dominant hamartomatous disorder with an incidence of approximately 1/6,000 (Hyman and Whittemore, 2000). Benign and occasionally malignant tumors occur in any organ, but predominantly the brain, kidney, heart, lung, and skin (Gomez, 1999). Mutations in either TSC1 or TSC2, encoding hamartin and tuberin, respectively, are the principle cause of TSC (Consortium, 1993; van Slegtenhorst et al., 1997). Although any organ can be affected, brain pathology, often as cortical tubers and subependymal nodules, is particularly debilitating. Significant morbidity and mortality result from epilepsy, autism, and other developmental and behavioral disabilities. TSC1 and TSC2 control cell growth, division, adhesion, and vesicular traffic by modulating the insulin signaling/ AKT/mTOR, Wnt/Disheveled/b-catenin, and Rho-GTP/ p27 Kip1 pathways (Au et al., 2004). How perturbations in these pathways lead to pathology is unclear. Conventional targeted disruptions of the Tsc1 and Tsc2 genes in the mouse have been generated to model and study TSC (Kobayashi et al., 1999, 2001; Kwiatkowski et al., 2002; Onda et al., 1999; Wilson et al., 2005). Homozygous disruption of either gene causes embryonic lethality. Heterozygous mice eventually develop some aspects of TSC (renal tumors), but have not been demonstrated to have any major central nervous system pathology. To address CNS pathology, a conditional disruption of the Tsc1 gene has been crossed to a glial-specific Cre transgenic mouse (Jansen et al., 2005; Uhlmann et al., 2002). All of the progeny develop seizures after 1 month of age and demonstrate increased numbers of astrocytes as well as hippocampal disorganization. These Tsc1-null mice may be a good model for the neuropathology and seizures associated with TSC. To further study the CNS pathophysiology of TSC and better define the roles of Tsc2 in normal brain physiology, we have generated and characterized a conditional disruption of the Tsc2 gene using the Cre-loxP and Flp-frt systems. To generate a floxed Tsc2 allele, we made a Tsc2 neo targeting vector by inserting a loxp-bamhi site into exon 4 and a loxp-frt-neo-frt cassette into exon 1 (Fig. 1a). To screen for neomycin resistant ES cells we used the 3 0 external and 5 0 internal probes indicated. Seven of 180 clones demonstrated 13 kb wildtype and 8.6 kb mutant bands on Southern blot when the genomic DNA was digested with BamH1 and probed with the external probe (Fig 1b). After Southern analysis of BamHI digested DNA using the internal probe demonstrated the correct 13 kb wildtype and the 2.6 kb mutant bands (Fig. 1b), two of the clones were selected for blastocyst injection. Only one chimera demonstrated germline transmission as demonstrated by PCR genotyping for the presence of the loxp-bamh1 site (Fig. 1c). Breeding of Tsc2 þ/neo heterozygotes did not generate any live born Tsc2 neo/neo homozygotes, suggesting that the neomycin gene interfered with the expression of Tsc2. We dissected pregnant females from heterozygous Tsc2 þ/neo crosses at different embryonic time points * Correspondence to: Michael J. Gambello, Department of Pediatrics, Division of Medical Genetics, University of Texas Health Science Center, MSB 3.144, 6431 Fannin, Houston, TX michael.j.gambello@uth.tmc.edu Contract grant sponsor: Tuberous Sclerosis Alliance Published online in Wiley InterScience ( DOI: /dvg.20271

2 102 HERNANDEZ ET AL. FIG. 1. Generation of the Tsc2 neo allele. (a) Gene-targeting construct. Exons are indicated by black boxes labeled with exon numbers. LoxP sites are marked by black triangles and frt sites by white triangles. The loxp-frt-neo-frt cassette was inserted in intron 1 and a loxp-bamhi site in intron 4. External and internal probes used for ES cell screening are indicated. PCR primers P1F and P1R were used to confirm germline transmission of the Tsc2 neo allele. B, BamHI; A,AflII; Sf, SbfI. (b) Southern blot analysis of genomic DNA digested with BamHI from Tsc2 þ/þ and Tsc2 þ/neo ES cell clones and probed with the external probe (Probe A). Positive clones (Lanes 1, 3) demonstrate the shorter 8.6 kb band because of the introduced BamHI site. Hybridization using the internal probe (Probe B) shows 13 and 2.6 kb fragments for both Tsc2 þ/neo clones (Lanes 1,2). (c) PCR product from mouse DNA using Primers P1F and P1R. Lane 1 shows a heterozygote Tsc2 þ/neo with a 433bp loxp-bamhi allele and a 389bp wild type allele; Lane 2, homozygous Tsc2 neo/neo embryo DNA; Lane 3 wild type. [Color figure can be viewed in the online issue, which is available at and found that most Tsc2 neo/neo embryos died at midgestation (Fig. 2b, c). Often nongenotypable resorptions were found with every heterozygous cross analyzed. Most Tsc2 neo/neo embryos that we were able to genotype were smaller and paler than the wildtype or Tsc2 þ/neo embryos and one had an open neural tube (Fig. 2b). These Tsc2 neo/neo embryos are similar to the previously reported Tsc2 homozygous knockouts in that they are smaller and paler than wildtype or heterozygous embryos. They differ in that we never observed exencephaly in Tsc2 neo/neo embryos. Moreover we identified some Tsc2 neo/neo embryos at embryonic time points (13, 15, and 17 dpc) when, in previous reports, no homozygous Tsc2 knockout embryos were ever found (Kobayashi et al., 1999; Onda et al., 1999). We also observed that Tsc2 þ/neo heterozygous mice developed renal cysts after a year of age (Fig. 2d, e). We have not detected frank renal tumors in these mice. Reported Tsc2 knockout mice all developed renal cysts and tumors within a year of life. These genetic data suggest that the Tsc2 neo allele is hypomorphic. To confirm this biochemically, we performed western analysis of whole embryo lysates (Fig. 2a.) Strong tuberin bands are seen for the wild type and the Tsc2 þ/neo lysates but a faint tuberin band is visible for Tsc2 neo/neo. Note that hamartin levels are unaffected and there is an increase in phosphorylated S6 in both Tsc2 þ/neo and Tsc2 neo/neo lysates, indicating activation of the mtor pathway. These data demonstrate genetically and biochemically that the Tsc2 neo allele is hypomorphic. To generate a conditional allele of Tsc2 that would be useful for modeling the multiorgan pathology of TSC in the mouse, we removed the neomycin gene by mating the Tsc2 þ/neo to FLPe transgenic mice (Fig. 3a) (Rodriguez et al., 2000). We confirmed that the neomycin gene was removed by PCR detection of the loxp-frt site that remains after Flp recombination (Fig. 3b). The resultant Tsc2 þ/flox mice were intercrossed to generate Tsc2 flox/flox homozygotes, which are viable, fertile and appear to behave like wildtype mice. To further characterize the Tsc2 flox/flox mice and ensure their utility for future organ and cell specific Cre-mediated deletion, we perfused mice with 4% paraformaldehyde and performed routine histologic analysis of various organs. No differences were found in the cerebral cortex, hippocampus, cerebellum, liver, lung or kidneys of the Tsc2 flox/flox mice when compared with the littermate control (Fig. 4). To demonstrate that the Tsc2 flox allele could be converted to a null allele by Cre recombination, we mated the Tsc2 flox/flox mice with a CMV-Cre transgeneic mouse (Fig. 3a). Complete deletion of exons 2 4 was demonstrated by PCR genotyping (Fig. 3c). To demonstate genetically that our Tsc2 ko allele is truly a null allele, we

3 CONDITIONAL DISRUPTION OF THE Tsc2 GENE 103 FIG. 2. Analysis of Tsc2 neo allele (a) Western analysis of lysates from E12.5 embryos. Note the low level of tuberin antigen in the Tsc2 neo/neo lane. Hamartin levels are unaffected. Phosphorylated S6 is increased in both Tsc2 þ/neo and Tsc2 neo/neo lysates. (b) Comparison of embryos at E12.5. Tsc2 neo/neo embryos were slightly smaller than wildtype and were distinctly underdeveloped and pale. Most noticeable is the lack of digitation of paws and the head size. Note the open neural tube indicated by the arrows. (c) Genotype frequency of Tsc2 þ/neo crosses demonstrating the lethality of Tsc2 neo/neo embryos. Asterisk (*) denotes nongenotypable resorptions. (d) Kidneys from Tsc2 þ/neo mice at various ages demonstrating cysts, and an H and E histology of a simple cyst, demonstrating renal cyst. Bar represents 1 mm for gross kidney photographs. (e) Table characterizing the kidney cyst phenotype. Note that no cysts developed in the first year of life, most cysts were 1 mm or less. No tumors were detected. [Color figure can be viewed in the online issue, which is available at wiley.com.] performed heterozygous crosses to determine if our homozygous knockout Tsc2 ko/ko die at midgestation as previously reported. No adult homozygous knockout Tsc2 mice were detected (Fig. 5c). Embryonic analysis at midgestation demonstrated midgestation lethality similar to previously published reports. Severe growth retardation was noted in the Tsc2 ko/ko mice (Figure 5b). Western analysis of whole embryo lysates demonstrates the complete absence of tuberin antigen in the Tsc2 ko/ko embryos, with unaffected hamartin levels (Fig. 5a). Note in particular that phosphorylated S6 is increased in both Tsc2 þ/ko and Tsc2 ko/ko embryos, demonstrating the activation of the mtor pathway. The kidney phenotype in our Tsc2 þ/ko mice is also similar to previously published reports. Lesions are detected within the first year of life (Fig. 5h) and are comprised of cysts and frank tumors (Fig. 5d,e). Histology demonstrates cystadenomas and renal tumors with abnormal giant cells (Fig. 5f,g). Though TSC is complex, efforts to understand the pathophysiology of this disease have been rather fruitful. TSC2 occupies a central role in inhibiting mtor activity (Au et al., 2004). Loss of tuberin results in increased mtor activity with consequent increases in translation, cell growth, and proliferation. Rapamycin, a well known inhibitor of mtor, has been used in trials to replace the TSC2 inhibition lost in TSC patients. Animal and human trials have been very encouraging (Franz et al., 2006; Kenerson et al., 2005; Lee et al., 2006). Nonetheless, tumors can often develop resistance to one chemotherapeutic agent. Consequently other targets should be

4 104 HERNANDEZ ET AL. FIG. 3. Generation of the Tsc2flox and Tsc2ko alleles. (a) The Tsc2neo allele was converted to a floxed allele by mating with a FLPe transgenic mouse. Heterozygous and homozygous Tsc2flox mice were obtained at the expected Mendelian frequencies, and homozygous mice were viable and fertile. A Tsc2ko allele was generated by mating the Tsc2flox mice with a general Cre-deletor strain. (b) PCR of tail DNA from conditional knockout mice using primers P2F and P2R. The 1172bp band indicates the presence of the loxp-frt site while the 1000bp band represents a wildtype allele. (c) PCR of tail or embryo DNA using primers P4F, P3R, and P3F to detect the wild type and the Tsc2ko alleles. The 1090 bp band represents the null allele. Homozygous Tsc2ko/ko genotypes were only identified at embryonic time points due to the lethality of the null phenotype (Lane 3). FIG. 4. Comparison of organs of wild type vs Tsc2flox/flox mice. Tsc2þ/þ: a, c, e, g, i, k; Tscflox/flox: b, d, f, h, j, l. (a, b) Cerebral cortex. (c, d) Hippocampus. (e, f) Cerebellum. (g, h) Liver. (i, j) Lung. (k, l) Kidney. Magnification: 403 (a d); 2003 (e l). sought by further understanding the pathophysiology of TSC, particularly in the brain. The conditional allele of Tsc2 that we have created is susceptible to Cre-mediated deletion to form a null allele. This allele will be valuable for studying postnatal organ and cell-specific pathology. Such postnatal studies will expand our knowledge of TSC pathology and shed new light on novel functions of TSC2.

5 CONDITIONAL DISRUPTION OF THE Tsc2 GENE 105 FIG. 5. Analysis of Tsc2 ko allele. (a) Western analysis of E12.5 embryo lysates. Note the absence of tuberin in Tsc2 ko/ko lysates, with the preservation of hamartin levels in all genotypes. Phosphorylated S6 is increased in Tsc2 þ/ko and even more so in the Tsc2 ko/ko lysates, demonstrating activation of the mtor pathway. (b) Comparison of embryos at E12.5. Wildtype and Tsc2 þ/ko mice are virtually identical. The Tsc2 ko/ko mouse is severely growth retarded. (c) Genotype frequency of Tsc2 þ/ko crosses demonstrating the lethality of Tsc2 ko/ko. Asterisk (*) denotes non-genotypable resorptions. (d) Kidney phenotype. Note the multicyst development (white arrows) in Tsc2 þ/ko mice. A small tumor is indicated by the black arrowhead (e) A large tumor was found in a Tsc2 þ/ko mouse at 11 months of age. (f) Histology of a complex cyst. (g) Histology of tumor shown in panel e. Note the presence of giant cells and nuclei (white arrowheads). (h) Table characterizing the kidney phenotype. Note that cysts as well as tumors were detected within the first year of life, much sooner than with the Tsc2 neo phenotype. [Color figure can be viewed in the online issue, which is available at MATERIALS AND METHODS Generation of Targeting Construct The targeting construct was made by PCR amplification of genomic DNA (Jackson Laboratories). All cloning was performed in the pbluescript backbone (Stratagene). A 5 kb fragment containing exons 1a-2 was amplified using primers T2E forward AGAAGGCAG- CACCTCTGTCA and T2E reverse CCACCTCCTCAAGC- TTCTTA. The 3 0 arm containing exons 2 6 was amplified using T2F forward TATTATCGATGTGAGTGGCCTCTGT- CTTGG and T2F reverse TATTCCCGGGCAGTGCAGG- TAAGCGAGAGT and cloned into a Bluescript vector containing loxp-frt-neo-frt. AloxP and a new BamH1 site were introduced into the SbfI site in intron 4. Both vectors were then combined to generate the full length targeting vector. All exons and intron exon junctions were sequenced to ensure no deleterious mutations were introduced. The functionality of the loxp and frt sites were tested in bacteria containing Cre and Flp respectively (data not shown). Linearized plasmid was electroporated into R1 ES cells. Screening with an external probe yielded 7 positive clones out of 180. Two of these clones were selected for blastocyst injection and yielded high level mosaic mice, which were mated with albino C57BL/6 mice. Only one clone demonstrated germline transmission as evidenced by PCR genotyping. Genotyping Genomic DNA was isolated from tail snips using routine protease digestion and phenol:chloroform extraction. For Southern analysis, DNA was digested with BamHI, separated on a 1% agarose gel and transferred to nylon. Probes were labeled with high prime (Amersham) to high specific activity and hybridized using ULTRAhyb TM (Ambion). Washed nylon was exposed to Kodak XAR film at 808C. PCR primers were developed to detect all of the various alleles. To genotype the Tsc2 neo allele, primers P1F 5 0 -CAGGCATGTCTGGAGTCTTG-3 0 and P1R 5 0 -CAG- CAGGTCTGCAGTGAATC-3 0 were designed to detect the presence of the loxp-bamhi site introduced into intron 4 (Fig. 1a). To genotype the Tsc2 flox allele, primers P2F 5 0 -TCCGGCTTGAAGGAGAAGTT-3 0 and P2R 5 0 -ATTGTT- GAGGCCGCATTCAC-3 0 were designed to detect the loxp-frt sites that would remain after removal of the neomycin cassette by Flp recombination (Fig. 3a). Primers P3F 5 0 -AAGATTCCGGCTTGAAGGAG-3 0, P4F 5 0 -CACTA- GTCTAGCCTGACTCT-3 0, and P3R 5 0 -GAGGACAAGCCA-

6 106 HERNANDEZ ET AL. ACATCCAT-3 0 were designed to detect the null allele as well as the wildtype allele (Fig. 3a). Protein Analysis Whole cell lysates were made from E12.5 embryos that were quick frozen in liquid nitrogen. After genotyping a small piece of each embryo, samples were pooled by genotype and homogenized in a dounce homogenizer with 10 volumes of Ripa buffer with protease inhibitor cocktail (Sigma). Lysates were centrifuged at 48C, sonicated and frozen until use. Protein concentrations were determined with a BCA reagent kit (Pierce). Equal amounts of protein were separated on a denaturing 4 12% gradient gel (Invitrogen) and transferred to nitrocellulose. The same membrane was probed with three different antibodies using a stripping procedure after each experiment. The membrane was first probed with tuberin antibody (1:1,000, Cell Signaling), then with hamartin (1:1,000, Santa Cruz), and lastly with phosphorylated (Ser 240/244) S6 (1:2,000, Cell Signaling). Secondary antibodies were horseradish peroxidase conjugated. Visualization was done with an enhanced chemiluminescence kit (Amersham). Embryo and Organ Analysis The day of the vaginal plug was 0.5. Mice were then anesthetized with avertin and killed by cervical dislocation before embryos were dissected into cold PBS. The yolk sac or a small piece of the embryo was used for genotyping. For organ analysis, mice were anesthetized with avertin and transcardially perfused with 4% paraformaldehyde. Organs were postfixed in paraformaldehyde overnight and then dehydrated and embedded in paraffin. Five micron microtome sections were cut. Slides were then rehydrated, stained with routine hematoxylin and eosin, and coverslipped. Embryo and tissue images were captured with a SPOT RT digital camera. ACKNOWLEDGMENTS We thank Gail Martin for the loxp-frt-neo-frt construct. Special thanks to Jan Parker-Thornberg and Richard Behringer for reagents and use of the MD Anderson Core facility. 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