Inconsistency between hepatic expression and serum concentration of transthyretin in mice humanized at the transthyretin locus Blackwellrunning Inconsistent Authors Publishing TTRhead: expression IncG Zhao in ethumanized al. mice
Gang Zhao1, Zhenghua Li1, Kimi Araki1, Kyoko Haruna1, Kazuhito Yamaguchi2, Masatake Araki3, Motohiro Takeya4, Yukio Ando5 and Ken-ichi Yamamura1,* 1
Department of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan 2 Institute of Life Science and Medicine, Science Research Center, Yamaguchi University, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan 3 Division of Bioinformatics, Institute for Resource Development and Analysis, Kumamoto University, 2-2-1 Honjo,Kumamoto 860-0811, Japan 4 Division of Cell Pathology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo,Kumamoto 860-8556, Japan 5 Department of Diagnostic Medicine, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1Honjo, Kumamoto 8608556, Japan
Human transthyretin (TTR) has about 110 variants, more than 90 of which are associated with human amyloidosis. Several groups have generated transgenic mice that carry various mutant TTR genes. However, formation of mouse/human TTR heterotetramers has been shown to be inhibitory to dissociation and subsequent amyloid formation. To avoid the effect of mouse Ttr and produce humanized mice carrying different TTR variants at high efficiency, we first produced a null allele in the mouse transthyretin locus using targeting vector that contained a neomycin resistance gene flanked by lox71 and loxP. Then, through Cre-mediated recombination, we created a replacement allele that carried either a human normal (Val30) or mutant (Met30) TTR cDNA. This replacement resulted in a humanized TTR mouse with similar tissue-specific profile of human TTR as that of the endogenous mouse Ttr gene. The expression levels of human TTR mRNA and protein in the liver of homozygous human TTR (Val30/Val30) mice were about twice those of heterozygous mouse/human TTR (+/Val30) mice. However, the serum human TTR levels in the Val30/Val30 mice were much less than those in the +/Val30 mice. This contradictory expression was due to unstable Val30 tetramers caused by low binding affinity to mouse retinol binding protein.
Introduction Human transthyretin (TTR) is encoded by a single-copy gene on chromosome 18. The gene spans about 7 kilobases (kb) and contains four exons and three introns (Tsuzuki et al. 1985; Wallace et al. 1985). TTR is synthesized mainly in the liver, choroid plexus and retinal pigment cells, and is secreted into plasma, cerebrospinal fluid, and vitreous body, respectively (Liddle et al. 1985; Herbert Communicated by: Shinichi Aizawa *Correspondence:
[email protected]
et al. 1986; Cavallaro et al. 1990). It serves as a transport molecule for thyroxine (T4) and the retinol binding protein (RBP) and circulates as a tetramer (55 kDa) composed of four identical, non-covalently associated subunits (Vranckx et al. 1990; Schreiber 2002). Although each monomer (15 kDa) consists of 127 amino acid residues, about 110 TTR variants have been identified. More than 90 of these are associated with human amyloidosis. While the phenotypes vary in TTR-associated amyloidosis (ATTR), polyneuropathy and cardiomyopathy are the major clinical manifestations that result from the extracellular deposition of amyloid fibrils composed of mutant
DOI: 10.1111/j.1365-2443.2008.01242.x © 2008 The Authors Journal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
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forms of TTR (Andrade 1952). In addition to the amyloidogenic TTR variants, the wild-type protein itself can form amyloid fibrils in cases diagnosed as senile systemic or senile cardiac amyloidosis (SSA or SCA). The latter condition affects 20%–25% of people over the age of 80 years (Cornwell et al. 1983). To gain insight in the pathogenesis of TTR-associated amyloidosis (ATTR), several groups have generated transgenic mice that carry various mutant TTR genes, such as Met30, Ser10/Met30, Pro55, or Ser84 (Yi et al. 1991; Araki et al. 1994; Watts et al. 1996; Takaoka et al. 1997; Teng et al. 2001; Sousa et al. 2002; Reixach et al. 2007; Tagoe et al. 2007). Using the mouse metallothionin promoter or human homologous TTR promoter, we produced transgenic mice in which amyloid deposition was observed (starting at the age of 6 months) in similar tissues as in human autopsy cases, except for its absence in the peripheral and autonomic nervous systems (Yi et al. 1991; Takaoka et al. 1997). Teng et al. have also reported amyloid deposition in transgenic mice for the wild-type human TTR gene (Teng et al. 2001). In contrast, transgenic mouse strains with amyloidogenic Pro55 develop non-fibrillar TTR deposition, but fail to develop amyloid deposits. Later, Reixach et al. demonstrated that heterotetramers composed of mouse and human subunits are kinetically more stable than those of human homotetramers and are considered to be inhibitory to dissociation and subsequent amyloid formation (Reixach et al. 2008). Actually, Sousa et al. have reported the presence of amyloid fibrils in Pro55 transgenic mice with a TTR null background (Sousa et al. 2002). Conventional gene targeting has been very useful in the study of gene function and regulation in mice. However, it is laborious and time-consuming to make a targeting vector and select targeted ES clones. When faced with
the development of various mouse models with different human TTR gene variants to analyze their roles in pathological conditions, classical gene targeting meets an insurmountable barrier. Previously, we have successfully overcome this challenge by Cre-mediated recombination of mutant lox sites (Araki et al. 1997, 1999, 2002). Here, we succeeded to establish the humanized mice by isolating ES clones with the targeted null Ttr allele with mutant lox sites. These ES clones enabled us to efficiently knock-in a human normal (Val30) or mutant (Met30) TTR cDNA to mouse Ttr gene locus. We showed that the replaced human TTR cDNA is expressed in a similar pattern to the mouse endogenous Ttr gene in terms of tissue-specific expression. The expression levels of human TTR mRNA and protein in the liver of homozygous human TTR (Val30/Val30) mice were about twice as those of heterozygous mouse/human TTR (+/ Val30) mice. However, the serum human TTR levels in the Val30/Val30 mice were much less than those in the +/Val30 mice. Thus, humanized mice showed contradictory expression patterns of human TTR, high in the liver and low in the serum.
Results Establishment of ES clones and mouse strains with the targeted null allele or replaced allele of Val30 or Met30
The production of the replaced allele was done in two steps. The first step was the production of the targeted null allele by homologous recombination and the simultaneous introduction of lox71 and loxP sites (Fig. 1, middle panel). The second step was the Cre-mediated site-specific introduction of human Val30 or Met30 cDNA to
Figure 1 Creation of targeted and replaced alleles. Homologous recombination between the wild-type allele and the targeting vector resulted in the creation of a targeted null allele which carried the PGK-neo gene flanked by lox71 and loxP. In the second step, the targeted clones were electroporated with the replacement vector that carried the human TTR cDNA flanked by lox66 and loxP. Site-directed recombination occurred between lox71/lox66 and loxP/loxP, which resulted in the creation of the replaced allele. PCR primers, 2-20-1S, A9s, mTtr19, neo-F, neo-R, mTtr19 and SP-A, are shown.
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Figure 2 Selection of targeted ES clones and genotyping of humanized mouse. (A) Detection of targeted clones. ES clones in which the 3- and 9-kb bands were detected using a 5′ primer set (2-20-1S/lox71-PR) and 3′ primer set (A-9 s/mTtr6), respectively, were judged as the targeted clones (left panel). These clones were confirmed by the presence of a 6- and 4.7-kb band when digested with BamHI and XbaI, respectively, in Southern blot analysis (right panel). (B) Genotyping using tail DNA of knockout mouse. A wild-type and targeted allele gave 864- and 545-bp band, when amplified by primer set A-9s/m19 and neo-R/neo-F, respectively. A targeted allele gave 6- and 4.7-kb band when digested with BamHI and XbaI, respectively, in Southern blot analysis. (C) Genotyping using tail DNA of humanized mouse. A wild-type and replaced allele gave an 864- and 783-bp band, when amplified by primer set A-9s/m19 and A-9s/Sp-A, respectively. A replaced allele gave a 7.5- and 5-kb band when digested with BamHI and BglII, respectively, in Southern blot analysis.
produce the replaced allele (Fig. 1, lower panel). After electroporation with the targeting vectors, we isolated and analyzed 98 neo-resistant clones. These clones were screened for targeted recombination by long PCR and confirmed by Southern blot analysis. Five clones (Numbers 23, 26, 28, 29 and 41) showed the presence of the 3- and 9-kb products by long PCR using primers 2-20-1S/ lox71-PR and mTtr6/neoF, respectively, and the 6- and 4.7-kb bands by Southern blot analysis when digested with BamHI and XbaI, respectively, which indicated the presence of the targeted allele (Fig. 2A). Among five ES targeted clones, two (Numbers 26 and 28) showed germ-line transmission and these clones were therefore used in subsequent experiments. Replacement vectors that contained either Val30 or Met30 cDNA were electroporated into the targeted ES clones together with the Cre expression vector. Twelve puromycin-resistant colonies were isolated in each experiment and analyzed for the presence of the replaced allele. Surprisingly, all 12 puromycin-resistant clones in each experiment contained 783-bp bands derived from the replaced allele, as revealed by PCR analysis with primers A9-s/Sp-A. This was confirmed by the presence
of a 7.5- or 5.0-kb band by Southern blot analysis when digested with BamHI or BglII, respectively, using the puromycin probe. Genotyping of the knockout or humanized mice was done either by PCR or Southern blot analyses, as shown in Fig. 2B,C. The mouse wild-type allele gave an 864bp band when primer set A-9s/mTTr19 was used, while the targeted allele gave a 545-bp band when primer set neoR/ neoF was used (Fig. 2B). In Southern blot analysis with the neo probe, the targeted allele gave a 6- or 4.7-kb band when digested with BamHI or XbaI, respectively (Fig. 2B). Both the replaced allele with Val30 and Met30 gave a 783-bp band when primer set A-9s/Sp-A was used in PCR analysis. In Southern blot analysis with a puromycin probe, the replaced allele gave a 7.5- or 5.0-kb band when digested with BamHI or BglII, respectively (Fig. 2C). Humanized mice of all genotypes appeared normal at least up to 6 months of age. Tissue-specific expression of Val30 cDNA
To examine the tissue-specific expression of Val30 cDNA, RNA was extracted from various tissues and analyzed by
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Figure 3 Expression of human TTR in humanized mice. (A) Tissue-specific expression of the mouse TTR and Val30 gene in Northern blot analysis. (B) Immunohistochemical staining showed the expression of Val30 in choroid plexus. Scale bars, 100 μm. (C) The mRNA levels of human TTR in the liver were almost the same among Val30/Val30, Val30/Met30 and Met30/Met30 mice. In addition, the levels of human TTR protein in the liver and serum were the same among these mice.
Northern blotting. In all mice examined, the Val30 cDNA was consistently expressed in the liver, brain and eyes (Fig. 3A), which are precisely the same tissues in which the mouse endogenous Ttr gene is expressed (Wakasugi et al. 1986; Cavallaro et al. 1990). The ratio of Val30 mRNA in the liver and brain of Val30 mice was similar to that of mouse Ttr mRNA (Fig. 3A). To examine the cell-type specificity of TTR expression in the brain, immunohistochemical analysis was performed using rabbit anti-human TTR antibody that recognized human TTR protein but not mouse TTR protein. The human TTR was detected in choroid plexus of +/Val30 and Val30/Val30 mice, but not in wild-type mice (Fig. 3B). This suggests that the regulatory elements in the mouse Ttr gene can direct the correct tissue-specific expression of the Val30 cDNA located in the replaced allele.
−/−, +/+, +/Val30 and Val30/Val30 mice using Northern and Western blot analyses. As expected, both mouse and human mRNAs were detected in the liver of +/Val30, but only human mRNA was detected in the liver of the Val30/Val30 mice (Fig. 4A). The level of human mRNA in Val30/Val30 was twice that in +/Val30 mice. In accordance with this, the level of human TTR protein in the livers of Val30/Val30 was twice that in the livers of +/Val30 mice (Fig. 4B). These results showed human TTR were expressed in a gene-dose-dependent manner in these mice. However, the serum level of human TTR in Val30/Val30 mice was less than half of that in +/Val30 mice (Fig. 4C), which suggested that the low serum level of human TTR in the Val30/Val30 mouse was due to the post-translational event in liver cells. Histological analysis
Consistent expression of human TTR cDNA after replacement
We examined the consistency of human TTR cDNA expression from the replaced allele using Val30/Val30, Val30/Met30 and Met30/Met30 mice. The mRNA and protein levels of human TTR in the liver were almost the same among these mice (Fig. 3C). Thus, we can expect the same level of expression from the replaced allele and this system is quite useful for production of other strains of mouse carrying different mutations, such as Pro55 or Met119. Comparison of the expression of human TTR in liver and serum
We carried out detailed analyses for the expression levels of mRNAs and proteins of human and mouse TTR in 1260
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Although the serum levels of human TTR in humanized mice were low, they appeared normal at least up to 6 months of age and there was no histological abnormality in tissue sections. This is consistent with the data that Ttr knockout mice appeared normal in the complete absence of mouse TTR in the serum (Episkopou et al. 1993). Transmission electron microscopy
There are two possible reasons for the low serum level of human TTR in the humanized mice. One is impairment of secretion of human TTR in mouse liver. We carried out an electron microscopy study to examine whether Val30 homotetramers were retained in the secretory pathway. However, no morphological change
© 2008 The Authors Journal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Inconsistent TTR expression in humanized mice
the normal secretory pathway. We carried out immunofluorescent studies using a TCS-SP2 AOBS confocal microscope. Liver sections were double stained with anti-TTR antibody labeled with red fluorescence and anti-calnexin antibody labeled with green fluorescence or anti-GM130 with green fluorescence. TTR signals were found in both the ER and Golgi, as revealed by the overlap with calnexin and GM130 markers. There was no difference in distribution pattern among +/+, +/ Val30, and Val30/Val30 mice (Fig. 5B), which indicates that human TTR was efficiently trafficked through the normal protein secretory pathway. Monitoring pH-induced TTR tetramer dissociation
Figure 4 Comparison of the expression of human TTR in liver and serum. (A) Expression of mouse and human TTR mRNA in the liver. The expression level of Val30 mRNA in Val30/Val30 mouse was almost twice that in +/Val30 mice. (B) Expression of mouse and human TTR protein in the liver. The expression level of Val30 protein in Val30/Val30 mice was almost twice that in +/ Val30 mice. (C) Serum levels of mouse and human TTR. The serum level of Val30 protein in Val30/Val30 mice was lower than that in the +/Val30 mice. Black bars: mouse TTR; open bars: human TTR.
was observed in the secretory pathway, including the ER or Golgi apparatus in liver cells (Fig. 5A). Immunofluorescence microscopy
Calnexin and GM130 are markers for ER and Golgi apparatus, respectively, and we used these markers to follow the trafficking of human TTR protein through
Another possible explanation for the low serum level of TTR in the humanized mice was the low stability of human TTR homotetramers compared with that of mouse/ human TTR heterotetramers. Stability of tetramer was first analyzed using sera from +/+, +/Val30, Val30/Val30, Val30/Met30, and Met30/Met30 mice by examining the distribution of tetramers, dimmers, and monomers in sera from these mice using native ployacrylamide gel electrophresis (PAGE). The concentrations of tetramers in Val30/Met30 or Met30/Met30 mice were much less than that of tetramers in Val30/Val30 mice (Fig. 5C), suggesting that Val30/Met30 heterotetoramers and Met30 homotetramers are more unstable than Val30 homotetramers. Surprisingly, the concentration of dimmers in Met30/Met30 mice was higher than those in Val30/ Val30 or Val30/Met30 mice. As the concentration of tetramers in Val30/met30 and Met30/Met30 mice were very low, we could not examine the stability of tetramers in these mice, Thus, the stability of tetramers in Val30/ Val30 mice in comparison with that of tetramers in +/+ and +/Val30 mice was assessed by native PAGE. As shown in Fig. 5D, under acidic conditions (pH 4.0), mouse TTR homotetramers and mouse/human TTR heterotetramers were quite stable up to 4 days incubation. On the other hand, human Val30 homotetramers were dissociated easily and disappeared almost completely by 4 days incubation. The concentrations of human TTR monomers increased slightly concomitant with the decrease of tetramers up to 2 days incubation. Decrease of monomers after 2 days incubation might be due to conformational changes or degradation caused in acidic conditions. In addition, two tetramer bands were observed in Val30/Val30 mice. As only single band of Val30 homotetramers was observed under neutral pH, appearance of two bands may be due to conformational change of Val30 homotetramers following serum incubation in acidic condition.
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Figure 5 Analyses of contradictory expression of human TTR. (A) Electron microscopy of the liver from +/+, +/Val30 and Val30/ Val30 mice. No pathological change was found. Scale bars, 500 nm. (B) Immunofluorescence analysis using anti-TTR antibody and anticalnexin or anti-GM130 antibodies. Human TTR was present in the ER and Golgi. Scale bars, 10 μm. (C) Western blot analysis using native gel electrophoresis. Concentrations of tetramers in Val30/Met30 or Met30/Met30 mice are significantly lower than that of tetramers in Val30/Val30 mice. But, concentration of dimers in Met30/Met30 mice is higher than those in Val30/Val30 or Val30/met30 mice. (D) Western blot analysis by native PAGE using serum after incubation in acidic conditions. Human Val30 homotetramers were more unstable than mouse TTR homotetramers and mouse/human TTR heterotetramers. Among human TTR tetramers, Met30 tetramers were the most unstable and Val30/Met30 heterotetramers were more unstable than Val30 tetramers. Met30 dimers were more stable than Val30 dimers. t: tetramer; d: dimer; m: monomer. (E) Compared with the amount of Val30 in serum from +/Val30 mice, the amount of Val30 precipitated by anti-mouse RBP decreased obviously, suggesting inefficient binding of human TTR with mouse RBP. Black bars: mouse TTR; open bars: human TTR.
Immunoprecipitation experiment
We carried out immunoprecipitation experiments using both anti-mouse RBP and anti-human TTR antibodies. When we used anti-human TTR antibody, we did not obtain any result. This might be due to differences in binding affinity of antibody for complexed and non-complexed 1262
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form of TTR (Schweigert et al. 2006). Immunoprecipitation by anti-mouse RBP was successful and the data are shown in Fig. 5D. Compared with the amount of human Val30 in serum from +/Val30 mice, the amount of Val30 precipitated by anti-mouse RBP decreased obviously, suggesting inefficient binding of human TTR with mouse RBP.
© 2008 The Authors Journal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
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Figure 6 Serum levels of T3, T4, retinol and RBP. The serum levels of T3, T4, retinol and RBP in Val30/Val30 mice were lower than those in +/+, +/Val30 mice and were similar to those in –/– mice. The levels of retinol and RBP were more severely affected than those of T3 and T4, suggesting that other proteins, such as albumin and thyroglobulin, can bind to T3/T4 to maintain the serum levels. Data are presented as means ± SEM. *P < 0.01 (t-test) (n = 12).
Levels of plasma retinol, RBP and thyroid hormone
TTR plays an important role in plasma transport of both thyroid hormone and retinol, through binding to RBP. We determined T3, T4, retinol and RBP in sera from −/−, +/+, +/Val30 and Val30/Val30 mice. The serum levels of T3 and T4 in Val30/Val30 were slightly lower than those in +/Val30 and +/+ mice, but were similar to those in –/– mice (Fig. 6). On the other hand, the serum levels of retinol and RBP in Val30/Val30 were significantly lower than those in +/Val30 and +/+mice, but were similar to those in –/– mice (Fig. 6). These corresponded well to the low levels of TTR tetramers in humanized mice. However, the levels of retinol and RBP were more severely affected than T3 and T4, suggesting that other proteins, such as albumin and thyroglobulin, can bind to T3/T4 to maintain the serum levels.
Discussion In this study, we established new humanized mouse strains with the normal (Val30) or mutant (Met30) human TTR gene, with similar tissue-specific profile as that of the endogenous mouse Ttr gene. Interestingly, humanized mice showed contradictory expression patterns of human TTR protein, high in the liver and low in the serum. The Cre/loxP recombination system has proven to be the most useful tool for genetic manipulation of mammalian cells and mice (Lakso et al. 1992; Rossant & Nagy
1995). We previously developed the exchangeable gene trap method and showed that a marker gene flanked by the left element (LE) mutant lox (lox71) and loxP in the mouse genome can be efficiently replaced with a gene of interest flanked by the right element (RE) mutant lox (lox66) and loxP in a vector, through recombination between lox71/lox66 and loxP/loxP. Recombination between lox71 and lox66 produces a wild-type loxP site and a LE + RE mutant site that is poorly recognized by Cre, which results in stable integration (Araki et al. 1999, 2002). In this experiment, lox71 and loxP sites in the mouse targeted allele were recombined with lox66 and loxP sites in the replacement vector, respectively, which resulted in replacement of PGK-neo with a human TTR cDNA. Since the binding affinity of the lox71/66 site for Cre recombinase was reduced, the integrated β-globin-human TTR cDNA-polyA-FRT-pGK-puroFRT cassette was retained stably. Actually, in all of the clones, site-specific recombination occurred, which resulted in integration of human TTR gene. Thus, once a targeted allele that contained a set of lox71 and loxP sites was constructed, we could easily establish a new mouse strain that carried the human TTR gene with a desired mutation. As more than 90 TTR variants are associated with human amyloidosis and, our system is quite useful to produce many different humanized mouse strains to analyze, for example, tetramer stability, such as Met30/Thr119 or Met30/His104 heterotetramers (Coelho et al. 1996; Longo Alves et al. 1997; Terazaki et al. 1999).
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Human TTR cDNA in the replaced allele is expressed in the same tissues, including the liver, choroid plexus and eyes, as those in which the mouse endogenous Ttr gene is expressed. These results suggest that the promoter/enhancer of the mouse Ttr gene can direct the correct tissue-specific expression of inserted human TTR cDNA in the replaced allele, even though the insertion site is located in the first exon near to the transcription initiation site. It was surprising that the serum levels of human TTR in Val30/Val30 mice were much lower than those in +/ Val30 mice, although the levels of TTR mRNA and protein in the livers of Val30/Val30 mice were expressed in a gene-dose-dependent manner. We showed that this decreased serum level was mainly due to the low stability of human homotetramers in mice serum. Tagoe et al. (2007) demonstrated that the TTR tetramer is much more stable in the presence of the murine protein because the TTR circulates as mouse/human heterotetramers. Our results were consistent with these data. It was reported that small molecules, such as T4, RBP and Chromium ion, were shown to stabilize the TTR tetramer against dissociation and subsequent conformational changes required for amyloid fibril formation in in vitro experiments (Miroy et al. 1996; White & Kelly 2001; Sato et al. 2006). Together with the data from immunoprecipitation experiments, our results suggest that mouse RBP can hardly bind to human TTR and the instability of tetramers is due to the loss of binding of human TTR to mouse RBP. The molecular interactions between TTR and RBP molecules have been studied in detail (Monaco 2000; Newcomer & Ong 2000), and these studies have shown that amino acid residues are involved in the interaction between the two molecules. TTR amino acids that participate in the interaction, such as Val20, Arg21, Gly 83, Ile84 and Tyr114 (Monaco 2000) are conserved between humans and mice. In addition, there are no significant differences between the crystal structures of murine and human homotetramers (Reixach et al. 2008). In contrast, the carboxyl terminus of RBP, which appears to modulate RBP : TTR binding affinity (Newcomer & Ong 2000), was different in four out of eight amino acids (GRSERNLL in humans versus SRPSRNSL in mice). In fact, the naturally occurring loss of C-terminal Leu or Leu–Leu of RBP is more readily cleared from the plasma than full length RBP (Jaconi et al. 1995, 1996). Thus, differences in the C terminus may be responsible for low binding affinity of mouse RBP to human TTR. Almeida et al. have suggested that increased stability of the Met30/119Met heterotetramer is due to increased T4 binding affinity to TTR (Almeida et al. 1997). However, RBP is synthesized in 1264
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the liver and can form a complex with TTR in the ER before secretion (Bellovino et al. 1996), and the vast majority (> 99.5%) of the two T4 binding sites within TTR in both fluids are unoccupied (Stockigt 2001). Thus, RBP may play an important role in stabilizing TTR during secretion into serum when an expression level is low. Patients homozygous for the Met30 mutation have been shown to have milder symptoms than heterozygous patients (Holmgren et al. 1988). On the other hand, decreased stability of Met30 tetramers has been shown in several studies using recombinant proteins in vitro (Quintas et al. 1997; Nettleton et al. 1998; Niraula et al. 2002; Ferrao-Gonzales et al. 2003; Skoulakis & Goodfellow 2003). In accordance with these results, we found that Met30 homotetramers were more unstable than Val30/ Val30 homotetramers and Val30/Met30 heterotetramers. However, we found that Met30 dimers were more stable than Val30 dimers, suggesting that the presence of stable dimers was the reason for the milder phenotype in the Met30 homozygous patient. We observed decreased serum levels of T3/T4 and retinol/RBP in –/– mice. A similar finding was reported by Episkopou et al. (1993). In addition, serum levels of T3/T4 or retinol/RBP in Val30/Val30 mice were lower than those in +/+ or +/Val30 mice, but similar to those in –/– mice. These results corresponded well with the decreased levels of tetramers in Val30/Val30 mice. The serum levels of retinol/RBP were more severely affected than those of T3/T4 in Val30/Val30 mice. An explanation for this is as follows. T4 can bind to thyroglobulin and albumin, and these proteins are also carriers of T4 in plasma. On the other hand, TTR is the only transport molecule for RBP. RBP can form a complex with TTR in the ER before secretion (Bellovino et al. 1996) and circulates in plasma as a complex with TTR. Binding to TTR serves to prevent the loss of RBP from the circulation through glomerular filtration (Kato et al. 1984). In conclusion, following the disruption of mouse TTR gene and the introduction of mutant lox sites, we knocked in human TTR cDNA to the downstream of endogenous mouse TTR promoter through Cre-mediated recombination. The replaced human TTR cDNA is expressed in a similar pattern to the mouse endogenous Ttr gene in terms of tissue-specific expression. This technology enables us to produce humanized mice carrying any desired TTR variants at high efficiency. Thus, it is quite useful for production of humanized TTR mouse models to analyze factors involved in the pathogenesis of human ATTR and to devise a new way of treatment that can stabilize human TTR tetramers in vivo.
© 2008 The Authors Journal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
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Experimental procedures Construction of the targeting and replacement vectors The targeting vector for homologous recombination was designed to disrupt the expression of mouse Ttr gene, as well as introduce the lox71 and loxP sites into the mouse Ttr locus. A 3-kb 5′ homologous region upstream of the ATG codon and a 6.7-kb 3′ homologous region downstream of the ATG codon were ligated with a p71neoP cassette that contained lox71-PGK-neo-loxP-polyA, to produce the TTR71neoPTTR construct that comprised 5′ Ttr homologous region-lox71-PGK-neo-loxP-polyA-3′ Ttr homologous region. Finally, a diphtheria toxin A (DT-A) fragment with an MC1 promoter was ligated to the 5′ end of TTR71neoPTTR to produce the targeting vector (Fig. 1). For construction of the replacement vector, we first made a cassette that contained a rabbit β-globin intron II, a human normal TTR cDNA (Val30) or a human Met30 cDNA, and a puromycin resistance gene with the phosphoglycerokinase promoter (PGKpuro) flanked by two Flp recognition target (FRT) sequences. Then, the cassette flanked by lox66 and loxP sites was inserted into pSP73 (Promega, Tokyo, Japan) (Fig. 1).
Isolation of ES cells with the targeted null allele and replaced allele carrying either Val30 or Met30 The ES cell line TT2 was grown as described previously (Niwa et al. 1993). TT2 ES cells were electroporated (Bio-Rad Gene Pulser at 800 V, 3.0 μF) with 30 μg linearized targeting vector plasmid. The cells were plated on 10-cm plates. G418 (250 μg/mL, Sigma, St Louis, MO) was added to the culture media 24 h after electroporation. Neo-resistant colonies were picked at 8–10 days and transferred to 24-well plates. At confluence, half of the cells in each colony were plated on a gelatinized 12-well plate and re-expanded for PCR analysis and Southern blot hybridization. The rest of the cells were plated on a gelatinized 24-well plate to re-expand for frozen stock and production of chimeric embryos. The ES cell lines with the Ttr targeted null allele were coelectroporated (Bio-Rad Gene Pulser at 400 V, 125 μF) with 20 μg replacement vector plasmid and 20 μg pCAGGS-Cre plasmid (Araki et al. 1997) to produce ES clones with replaced allele. Here, puromycin was used for positive selection. The other procedures were similar to those for generation of ES clones with the targeted allele.
Generation of mouse TTR knockout or human TTR knock-in mice Chimeric mice were produced by aggregation of ES cells with eight-cell embryos from ICR mice, and then chimeric embryos were transferred into the pseudopregnant recipients. Chimeric male mice were backcrossed to C57BL/6 females (Nippon Clea, Kanagawa, Japan) and mice from more than the fifth generation were used in the following experiments.
PCR and Southern blot analyses for isolation of targeted ES clones The targeted ES clones were screened and confirmed by long PCR and Southern blot analyses using genomic DNA purified by phenol, chloroform and ethanol precipitation from ES cells. In long PCR analysis, a primer set, 2–20–1S (5′-GTAAGCAATCTTA GCCAGGCTCTCC-3′) and lox71-PR (5′-TATACGAACGGT ATAGGTCCCTCGAC-3′), was used to detect the 5′ end of the targeted allele (3.0 kb). A primer set, mTtr6 (5′-TGG GCT GAG TCT CTC AAT TCT G-3′) and neo-F (5′-AGAGGCTATTC GGCTATGAC-3′), was used to detect the 3′ end of targeted allele (9.0 kb). For Southern blot analysis, DNAs from ES cells were digested with BamHI or XbaI and were analyzed for the presence of the targeted allele using a neo probe. The digested DNAs were electrophoresed on a 1.2% agarose gel and blotted onto nylon membranes (Hybond-N+; Amersham, Tokyo, Japan). After the membranes were cross-linked by exposure to ultraviolet light (UV Stratalinker 1800; Stratagene, La Jolla, CA), hybridization was performed using a neomycin-specific probe prepared by using a DIG DNA labeling and detection kit (Roche, Tokyo, Japan).
PCR and Southern blot analyses for genotyping of ES clones or mice Genotypes of ES cells and mice were determined by PCR analyses and confirmed by Southern blot analysis using genomic DNA from ES cell or tails. In PCR analysis, a primer set, mTtr A9-s 5′CGTAGAGCGAGTGTTCCG-3′ and mTtr19 (5′-CAGCTGT TGCTATAGTAATTCCC-3′), was used to detect the wild-type mouse Ttr allele (864 bp). A primer set, neo-R (5′-CACCATGA TATTCGGCAAGC-3′) and neo-F (5′-AGAGGCTATTCGGC TATGAC-3′), was used to detect the targeted allele (545 bp). A primer set, A9-s and SP-A (5′-CAGTGTATATCATTGTAACC-3′), was used to detect the replaced allele (783 bp). For Southern blot analysis, 10 μg DNA from mice with the targeted allele and replaced allele were digested with BamHI or XbaI and BamHI or BglII, and were analyzed using a neo-specific or puromycinspecific probe, respectively.
Northern blot analysis For Northern blot analysis, total RNAs isolated from various tissues were electrophoresed on a 1.2% denaturing formaldehyde/MOPScontaining agarose gel and transferred to nylon membranes (Hybond-N+; GE, Tokyo, Japan). After RNAs were cross-linked by exposure to ultraviolet light, the membrane was prehybridized and then hybridized using the human TTR cDNA-specific RNA probes, the mouse TTR cDNA-specific probes, or the glyceraldehyde-3-phosphate dehydrogenase RNA probes prepared using DIG RNA labeling and detection kit (Roche).
Western blot analysis The liver was homogenized in lysate buffer [HEPES 50 mmol/L, pH 7.4, NaCl 150 mmol/L, Triton X-100 0.1%, glycerol 10%,
© 2008 The Authors Journal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
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G Zhao et al. NaF 1 mmol/L, sodium orthovanadate 2 mmol/L, EDTA 1 mmol/L, and a protease inhibitor cocktail (1 : 100 dilution); Sigma– Aldrich, St. Louis, MO]. The extracts (60 μg protein per lane) and mouse serum (6 μL mouse serum diluted 1 : 50 in 0.9% NaCl per lane) were applied to 12% SDS-PAGE and transferred to an immobilon polyvinylidene difluoride filter (Millipore, Billerica, MA). Primary antibodies were used at the indicated dilutions: rabbit anti-human TTR antibody (diluted 1 : 1000; MBL, Nagoya, Japan), rabbit anti-bovine serum albumin (BSA) antibody (diluted 1 : 1000; Upstate, NY), rabbit anti-β-actin antibody (diluted 1 : 1000) (Sigma). An anti-rabbit immunoglobulin G antibody conjugated with horseradish peroxidase (Amersham Biosciences, Piscataway, NJ) was used for detection. The intensity of the bands was quantified by densitometry using ImageJ software (version 1.38, a program inspired by NIH image; ).
Histological analysis For histological analysis, tissues including brain, heart, lung, alimentary tract, liver, spleen, pancreas, kidney, testis, lymph node, skeletal muscle, and skin were dissected out from mice and were fixed overnight in 10% formalin, embedded in paraffin, sectioned, and stained with the H&E.
Electron microscopy All animals were perfused with 5 mL of a 2% glutardehyde/2% paraformaldehyde mixture in 0.1 m cacodylate buffer (pH 7.4) via the thoracic aorta. The right atrium was cut to allow blood to flow out and perfusion with fixative. After perfusion, the same site of the right liver lobe from each animal was removed for examination and cut into 1-mm3 blocks for further fixation with the same fixative for 4 h at 4 °C. The liver blocks were post-fixed with 2% osmic acid, dehydrated with a graded series of acetone, and embedded in Epon 812. Ultrathin sections (90 nm) were cut with a Reichert Ultracut J using a diamond knife. The sections were stained with uranyl acetate and lead citrate and examined with a Hitachi H7500 electron microscope (Hitachi, Tokyo, Japan) at 80 kV.
Immunohistochemistry and immunofluorescence analysis Mice were killed after anesthesia with ether. Liver and brain were excised. The tissues were fixed in 4% paraformaldehyde in PBS, and embedded in paraffin for immunohistochemistry, or in OTC compound for immunofluorecent analysis. Immunohistochemistry was performed by using rabbit anti-human TTR antibody (diluted 1 : 200) (Abcam, Cambridge, UK). Although many anti-human TTR antibodies react with both human and mouse TTR, we confirmed that this antibody was very specific to human TTR by Western blot assay. Thus, we used this for detection of human TTR in mouse tissues. Primary antibody was detected with biotinylated anti-rabbit secondary antibody (diluted 1 : 200; Vectastain ABC Kit Rabbit IgG; Vector Laboratories, Burlingame, CA) and DAB detection kit (Ventana Medical Systems, Tucson, AZ)
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according to the manufacturer’s instructions. For immunofluorescent analysis, the frozen sections were incubated with mouse anticalnexin (BD Biosciences, San Diego, CA), mouse anti-GM130 (BD Biosciences), or rabbit anti-TTR (DAKO, Kyoto, Japan) to localize the endoplasmic reticulum (ER), Golgi apparatus or TTR, respectively. For visualization, the following secondary antibodies were used: anti-mouse Alexa Fluor488 or anti-rabbit Alexa Fluor594-conjugated antibodies (Molecular Probes, Eugene, OR). The nuclei were stained with 4′-6-diamidino-2′phenylindole (DAPI) (0.1 μg/mL; Sigma–Aldrich, Tokyo, Japan). Stained cells were observed using a TCS-SP2 AOBS confocal microscope (Leica Micro-systems, Wetzlar, Germany).
pH-induced TTR tetramer dissociation To determine the distribution of tetramers, dimmers, and monomers in sera from Val30/Val30, Val30/Met30, or Met30/Met30 mice, sera were diluted with six times volume of native sample buffer (0.05 m Tris–HCl at pH 6.8, 10% glycerol) and 6 μL of sample per lane was analyzed by 4%–20% gradient Tris–Glycine Gel (Invitrogen, Carlsbad, CA). To determine the stability of tetramers, 10 μL of mouse serum from +/+, +/Val30, and Val30/Val30 was added to 240 μL of 100 mm acetate solution (pH 4.0) containing protease inhibitor cocktail (1 : 100 dilution) and incubated at 37 °C for 3, 6, 12, 24 and 36 h, and 2, 3 and 4 days. These samples were diluted with same volume of sample buffer (0.05 m Tris–HCl at pH 6.8, 10% glycerol) and unboiled 6 μL of sample per lane was analyzed by 4%–20% gradient Tris–Glycine Gel (Invitrogen). Rabbit anti-human TTR antibody (diluted 1 : 1000; MBL) was used as primary antibody.
Co-immunoprecipitation For co-immunoprecipitation, rabbit anti-human TTR antibody (MBL) or mouse anti-RBP antibody (Abcam, Cambridge, USA) was pre-coupled to Dynabeads protein G (Dynal Biotech ASA, Oslo, Norway), followed by incubation with the sera of +/Val30 mice to immunoprecipitate the TTR–RBP complex according to manufacturer’s protocol. The immunoprecipitates were analyzed by Western blotting using mouse anti-RBP or rabbit anti-human TTR antibody.
Determination of serum triiodothyronine (T3), T4, retinol and RBP Serum T3, T4, retinol and RBP concentrations were measured commercially by radioimmunoassay (SRL Inc., Tokyo, Japan) when the mice were 12 weeks of age.
Acknowledgements Authors thank Kaoru Nagumo and Michiyo Nakata for their technical assistance. This study was supported in part by Grant-inAid for Scientific Research (A) from the Japan Society for the Promotion of Science (JSPS) and a grant from the Osaka Foundation of Promotion of Clinical Immunology.
© 2008 The Authors Journal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Inconsistent TTR expression in humanized mice
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