Glycobiology Advance Access originally published online on October 17, 2007
Glycobiology 2008 18(1):9-19; doi:10.1093/glycob/cwm114
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Production and Characterization of Transgenic Mice Systemically Expressing Endo-β-Galactosidase C
2 Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan
3 Division of Resources Life Science, United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-Minami, Tottori, 680-8553, Japan
4 Department of Biological Science, Faculty of life and Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane, 690-8504, Japan
5 Department of Organ Regeneration, Graduate School of Medicine, Shinshu University 3-1-1 Asahi, Matsumoto 390-8621, Japan
6 Department of Health Science, Faculty of Psychological and Physical Sciences, Aichi Gakuin University, 12 Araike, Iwasaki-cho, Nisshin, Aichi, 470-0195, Japan
7 Frontier Science Research Center, Kagoshima University, Korimoto 1-21-40, Kagoshima 890-0065, Japan
1 To whom correspondence should be addressed: Tel and Fax: +81-29-838-8662 e-mail: kettle{at}affrc.go.jp
Received on March 1, 2007; revised on September 26, 2007; accepted on October 5, 2007
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Abstract |
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The
Gal epitope (Gal1-3Gal) is a sugar structure expressed on the cell surface of almost all organisms except humans and old-world-monkeys, which express natural anti-Gal antibodies. The presence of these antibodies elicits a hyper acute rejection (HAR) upon xenotransplantation of cellular materials, such as from pigs to human beings. Endo-β-galactosidase C (EndoGalC), an enzyme isolated from Clostridium perfringens, removes the Gal epitope by cleaving the Galβ1-4GlcNAc linkage in the Gal1-3Galβ1-4GlcNAc sequence. To explore the possibility that cells or organs from transgenic pigs systemically expressing EndoGalC might be suitable for xenotransplantation, we first introduced the EndoGalC transgene into the mouse genome via pronuclear injection. The progeny of the resulting transgenics expressed EndoGalC mRNA and protein. Flow cytometry and histochemical analyses revealed a dramatic reduction in the expression of the Gal epitope in these mice. They also exhibited abnormal phenotypes, such as occasional death immediately after birth, growth retardation, and transient skin lesions. Interestingly, the phenotypic abnormalities seen in these transgenics were similar to those observed in β1,4-galactosyltransferase 1 (β4GalT-1) knockout (KO) mice. Most probably, these phenotypes were caused by exposure of the internal N-acetylglucosamine residue at the end of the sugar chain on the cell surface. The present findings also provide some basis for evaluating possible application of the transgenic approach for xenotranplantation.
Key words:
Gal epitope
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galactosidase lectin
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systemic promoter
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transgenic mice
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Extensive studies have demonstrated that glycan chains exhibit a broad array of functions in intercellular recognition and regulation of cellular activities (Alberts et al. 2002
; Lowe 2002; Partridge et al. 2004; Han et al. 2005; Ohtsubo and Marth 2006). Production of knockout (KO) mice lacking glycosyltransferase genes has been recognized as a powerful tool for elucidation of the role of glycan chains in glycoproteins and glycolipids (Lowe and Marth 2003). Overexpression of glycosyltransferases in cultured cells or transgenic mice has also yielded interesting results (Miyoshi et al. 1999; Sato et al. 2001; Wang et al. 2001). In this report, we analyze EndoGalC transgenic mice. This is the first report to analyze the effects of overexpression of a glycosidase as a tool to evaluate the biological roles of cell-suface glycans. This approach will complement the knockout approach in such cases where another member of the glycosyltransferase family is present that can compensate for the loss of function of the transferase that has been knocked out.
The Gal (Gal1-3Galβ) epitope is a sugar structure found in glycolipids and glycoproteins and is widely conserved in most organisms from bacteria to mammals (Zielenski and Koscielak 1982; Basu et al. 1996). The synthetic pathway of this sugar chain has been partially elucidated. In mammalian cells, enzymes of the β1,4-galactosyltransferase (β4GalT) family mediate addition of a galactose residue either to N-acetylglucosamine to form Galβ1-4GlcNAc-R or to glucose to form Galβ1-4Glc-R. Alpha1,3-Galactosyltransferase (3GalT-1) subsequently adds a galactose residue to the nonreducing end of Galβ1-4GlcNAc-R to form Gal1-3Galβ1-4GlcNAc-R (Blanken and Van den Eijnden 1985). Recently, another 1,3-galactosyltransferase (3GalT-2) has been shown to act on the synthesis of Gal epitope; so far, this enzyme has been reported to add galactose residue to the nonreducing end of the sugar chain on glycolipid (Taylor et al. 2003). However, human and old-world monkey cells do not express the Gal epitope, since the 3GalT-1 gene is inactivated in these cells (Lanteri et al. 2002). These animals possess natural antibodies against the Gal epitope, which leads to antibody-mediated rejection of xenotransplanted material, called the "hyper acute rejection (HAR)" (Cooper 1998). Mice and pigs that lack 3GalT-1 expression have been produced (Tange et al. 1996; Thall et al. 1996; Dai et al. 2002; Lai et al. 2002). A residual level of the Gal epitope remains in the 3GalT-1-deficient animals, and is thought to be produced by 3GalT-2 (Taylor et al. 2003; Milland et al. 2006). Mice deficient in 3GalT-1 exhibit no phenotype other than susceptibility to cataracts (Eyssens 1999). Whether this is due to compensation by 3GalT-2 is not known.
Endo-β-galactosidase C (EndoGalC), an enzyme initially isolated from the culture medium of Clostridium perfringens, can remove the Gal1-3Gal disaccharide by cleaving the Galβ1-4GlcNAc linkage (Fushuku et al. 1987; Ogawa et al. 2000). This enzyme does not cleave linkages containing residues other than -galactose, i. e., Sia2-3Galβ1-4GlcNAc-R or the linkage in the polylactosamine structure, (Galβ1-4GlcNAc)n (Fushuku et al. 1987; Muramatsu 1989). For this reason, EndoGalC is considered to be an effective tool for enzymatic removal of the Gal epitope from the surface of animal cells such as porcine cells. For example, Ogawa et al. removed most of the Gal epitopes from porcine kidney by perfusion of the organ with recombinant EndoGalC (Ogawa et al. 2000). Furthermore, EndoGalC expressed after gene transfer effectively removed the Gal epitope from pig endothelial cells (Ogawa et al. 2002), and introduction of the same gene into the rat transiently by an osmotic method was also effective to eliminate the Gal from hepatocytes (Miki et al. 2004). These results suggest that a transgenic approach to express EndoGalC could be useful for removing the Gal epitope so as to explore its biological function in living animals.
In this study, we produced EndoGalC-overexpressing transgenic mice using a strong and ubiquitously active chicken β-actin-based promoter. The progeny of these transgenic mice exhibited several abnormal phenotypes, the basis of which we discuss here.
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Generation of transgenic mice
We introduced an EndGalC expression vector (Ogawa et al. 2002
; Figure 1A) into the nuclei of fertilized mouse embryos to create transgenic mice. Of a total of 274 injected embryos, 237 (86.5%) survived and were transferred to the oviducts of 11 foster mothers. Twenty-three pups were born by natural birth and 43 were delivered by caesarean section. Of a total of 66 pups (27.8% of the transferred embryos) delivered, 58 survived until weaning and were subsequently genotyped for the presence of transgenes. Of the 58 mice, 6 (10.3%) founders were identified as transgenic. Three of these founders (termed TG-4, -5 and -10, expressing 1, 3 and 
30, copies of the transgene, respectively) (Figure 1B) appeared normal throughout their lives. However, the other three founders exhibited abnormal phenotypes, such as edema and dried skin and died shortly after birth. No overt anatomical abnormality was noted when their internal organs were inspected (data not shown).
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The TG-4, -5 and -10 founder mice were mated with C57BL/6 mice to obtain F1 progeny. However, the TG-4 founder (female) failed to produce any progeny despite repeated mating, probably due to sterility. Both TG-5 and TG-10 founders were fertile with an average litter of 7.
Semilethality of EndoGalC transgenic mice
The progeny of EndoGalC transgenic lines (TG-5 and TG-10) crossed with Wild-type (Wt) C57BL/6 mice were born healthy. The ratio of transgenicity in each line of mice was about 50%. This suggests that EndoGalC transgenic fetuses exhibit normal embryonic development. However, transgenics from both lines appeared to be slightly smaller than their nontransgenic littermates at birth. The body weight of the transgenics was 90–95% of that of their nontransgenic littermates (Figure 2B). Although the difference between the rate of growth of transgenics of the line TG-10 and their nontransgenic littermates was not evident until 6 days after birth, the transgenics did exhibit growth retardation (Figure 2B) around 7–12 days after birth and often died during this period (Figure 2A). All of the newborn mice appeared normal immediately after birth (Figure 2C), although skin lesions appeared around 7–12 days after birth (Figure 2D). They usually recovered around 10–12 days after birth, although traces of the lesions remained on their skin (arrows in Figure 2E and F). Pups that survived beyond the critical stages (7–12 days after birth) gradually exhibited normal growth rates and an overall normal appearance (Figure 2B). The phenotype of TG-5 mice was similar to that of TG-10 mice. TG-5 mice also exhibited skin lesions, although the lethality of TG-5 was lower than that of TG-10 (data not shown).
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Skin abnormality of EndoGalC transgenic pups during early postnatal development
Histological examination of transgenic pups 7–8 days after birth revealed that the epidermis was thickened at the affected area compared to that of their nontransgenic littermates (indicated by arrowheads in Figure 3B versus
D). Furthermore, the thickness of the paniculus adiposus was reduced in the transgenic mice compared to the nontransgenic littermates (indicated by asterisks in Figure 3A and C). Many keratohyalin granules were evident in the granular cell layers of the transgenic mice (arrows in Figure 3D). These findings indicate that the skin lesions in the EndoGalC transgenic pups resemble acanthosis and hyperkeratosis. As described above, most of the skin lesions disappeared 10–12 days after birth.
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Analysis of EndoGalC mRNA in pups and adults
Transgene expression was assessed using Day 8 transgenic pups from the TG-10 line and adult mice to correlate the abnormal phenotype with expression of EndoGalC mRNA by Northern blotting. Young pups (8 days after birth) exhibited mRNA expression in almost all organs, although the level of expression varied among the organs tested. The liver, kidney, lung, intestine, and muscle exhibited a high degree of expression, while the spleen and skin did not (Figure 4A). Weak expression of EndoGalC mRNA in the skin was unexpected, since severe phenotypic alteration had been noted in this tissue, as mentioned previously. In adult mice, we observed strong transgene expression in the cerebellum, kidney, and skin (where no skin lesion was noted), and weak expression in the liver and spleen (Figure 4B). The progeny of line TG-5 exhibited a similar pattern of expression of EndoGalC mRNA as the TG-10 line (data not shown). The TG-4 founder also expressed EndoGalC mRNA, although the level of expression appeared to be lower than that of the TG-5 and -10 lines (data not shown).
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Expression of EndoGalC protein and reduction of the expression of the
Gal epitope in the transgenic mouse cellsWe next examined EndoGalC protein expression in Day 8 transgenics by Western blot analysis (Figure 5A). The highest degree of expression was observed in the liver, with a moderate level of expression observed in the lung, muscle, and skin. Interestingly, an additional protein band of higher apparent molecular weight was detected in the intestine. A low level of EndoGalC protein was observed in the cerebrum, cerebellum, and kidney, while no expression was observed in the spleen. EndoGalC protein was not detected in the nontransgenic mice (Figure 5B). To determine whether expression of EndoGalC protein leads to reduced
Gal expression in mice, embryonic fibroblast cells (MEF) isolated from transgenic fetuses (Day 14) of the TG-10 line were stained with fluorescein isothiocyanate (FITC)-labeled GS-IB4 lectin and then analyzed by flow cytometry (Figure 5C). GS-IB4 is an isolectin isolated from Bandeiraea simplicifolia that specifically binds to nonreducing -galactosyl residue including the Gal epitope (Wood et al. 1979). We observed a 68% reduction in the level of staining by the lectin on the surface of MEFs in the transgenic mice. We next examined the degree of reduction in the Gal epitope using cells isolated from adult transgenic mice. Cells isolated from heart, kidney, lung, and pancreas were stained with FITC-labeled GS-IB4 lectin and then analyzed by flow cytometry. More than 90% of Gal expression was observed to be reduced in heart, kidney, and lung of the transgenic mouse, while only 40% reduction was noted in the pancreas (Figure 6A–D). These data indicated that EndoGalC could also be active in adult mice.
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Histochemical and immunohistochemical analyses of skin lesions and organs in the transgenic mice
To determine whether EndoGalC expression correlated with the transient skin lesions that appeared around 7–9 days after birth, we performed histochemistry and immunostaining using an anti-EndoGalC antibody and FITC-conjugated GS-IB4 isolectin on the skins of Day 8 transgenic pups. Immunostaining using the anti-EndoGalC antibody demonstrated that expression of EndoGalC protein was restricted to keratinocytes in the epidermis (arrowheads in Figure 7A) and the hair follicles (arrows in Figure 7A) in the transgenic mice. Nontransgenic skins did not exhibit reactivity to the antibody (Figure 7B). Histochemical staining with GS-IB4 isolectin revealed a significant reduction in reactivity in the skin cells of the transgenic mice (Figure 7A). In contrast, we observed staining for
Gal in most cells (except for the mesenchymal cells) of the nontransgenic mice (Figure 7B). A particularly high degree of staining was observed in the cells of the surface basal lamina (arrowheads) and in those surrounding the hair follicles (arrows). The skin of the 1-month-old transgenic mouse was also examined histochemically. EndoGalC expression was observed in all the epidermal cells (arrows in Figure 7C) in which reduced expression of Gal epitope was seen (arrows in Figure 7E). It was noted that Gal expression, which had not been seen in the 8-day-old pups, was observed in some epidermal cells (arrowheads in Figure 7D), in which expression of EndoGalC was greatly reduced (arrowheads in Figure 7E). These findings clearly indicate a reciprocal relationship between EndoGalC and Gal expression. Similarly, histochemical staining for the Gal epitope was performed using pancreas, lung, heart, and kidney tissue samples from a 21-day-old transgenic mouse one week after recovery of skin lesion (Figure 8). Expression of EndoGalC protein was observed in the pancreatic islet tissue including -cell and β-cell, which was critical for the secretion of hormones (arrows in Figure 8C and D). We found that expression of the Gal epitope was greatly reduced in these cells (closed arrowheads in Figure 8A and C). In addition, the terminal GlcNAc residue, which should be first exposed on the cell surface after EndoGalC digestion, was detected by the GS-II isolectin, a lectin known to specifically bind to the residue. However, distinct overlapping between EndoGalC expression and staining with GS-II isolectin was not seen (Figure 8B and D). The Gal epitope similarly disappeared from the transgenic lung, whereas its expression was slight in the nontransgenic mouse (closed arrowheads in Figure 8E and G), as reflected in the positive staining with GS-II isolectin seen in the transgenic lung, but not in the nontransgenic lung (Figure 8F versus H). The expression level of EndoGalC in the transgenic lung was low (arrows in Figure 8G), in contrast, to the relatively high expression of EndoGalC in the transgenic heart and almost all cardiac cells (closed arrows in Figure 8K), which were positively stained by GS-II isolectin (open arrowheads in Figure 8L). The Gal epitope had disappeared in this tissue (Figure 8K versus I). There was overlap between EndoGalC expression and staining with GS-II isolectin, although the signal for GS-II staining was patchy in the nontransgenic heart (open arrows in Figure 8L). In the kidney, no clear difference was seen between the nontransgenic and transgenic mice in staining with both lectins (Figure 8M–P). Although we did not isolate the β-cells from the pancreatic islet tissue and the endothelial cells, the specific Gal expression was not observed in these cells from the transgemic mice upon immunohistochemical analysis.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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In this study, we produced three lines of EndoGalC-overexpressing transgenic mice that exhibited reduced expression of the
Gal epitope. The EndoGalC transgenic mice expressed EndoGalC mRNA and protein in various major organs. However, we did observe a poor correlation between the levels of EndoGalC mRNA and protein within particular tissues. For example, whereas we detected strong expression EndoGalC protein in the skeletal muscle by Western blot analysis, the level of EndoGalC mRNA was relatively low as evaluated by Northern blot analysis (see Figure 4A). In contrast, in the kidney, the level of mRNA was relatively high, whereas protein expression was relatively low (see Figures 4A and 5A). We do not have an explanation for this phenomenon at the present time, but it may be related to the speed of posttranscriptional processing of EndoGalC mRNA, which appears to differ between organs. Interestingly, an extra protein band of larger apparent molecular weight than the 104-kDa EndoGalC protein was observed in the transgenic intestine (see Figure 5A), suggesting intestine-specific posttranslational modification of EndoGalC.
In this study, we have provided evidence that overexpression of EndoGalC abolishes the expression of the Gal epitope. Lectin staining demonstrated that nearly all skin cells from EndoGalC transgenic mice lost reactivity to the GS-IB4 isolectin (see Figure 6A). The Flow cytometry analysis provided quantitative data on the level of the remaining Gal epitope expression in MEF cells derived from transgenic fetuses as assessed by the reactivity to GS-IB4 isolectin. Gal antigen was reduced to 32% of that observed in the normal cells (see Figure 5C). These results clearly indicate that EndoGalC protein expressed in the transgenic mice was enzymatically active, as reported previously using cultured cells transfected with an EndoGalC expression vector (Ogawa et al. 2002), and following transient gene transfection in the rat (Miki et al. 2004).
Of the total of six founder mice obtained, three were unable to survive after birth because they had severe phenotypes, including edema and dried skin, that resulted in loss of moisture from the skin surface and finally lead to cracking of the skin in the neonatal stage. Such dried skin has been often observed in mouse mutants showing barrier dysfunction (Roop 1995; Rossant and Tam 2002). In many cases, these skin-related mutations are caused by a defect in the genes coding for the proteins functioning as signal transduction or as a component of extracellular matrix. Such genes are generally known to play a role not only in barrier function, but also during embryogenesis. EndoGalC might digest the critical sugar chains linked to these molecules and as a result, cause severe phenotypes in later development of embryos. Progenies of the survived EndoGalC-transgenic founder mice also showed some phenotypic abnormalities, such as postnatal semilethality, transient growth retardation, and skin lesions in newborn pups. Some transgenic mice surviving beyond the critical stage (7–9 days after birth) showed a "puffy face," probably reflecting hypothyroid myxedema (Jabbour 2003) (data not shown). These abnormal phenotypes found in the offspring of EndoGalC transgenic mice were similar to those observed in β1,4-galactosyltransferase-1 (β4GalT-1) KO mice (Lu et al. 1997). These latter mice exhibit growth retardation, frequent neonatal death, and other symptoms such as skin lesions. βGalT-1 is an enzyme capable of transferring galactose to the nonreducing end of N-acetylglucosamine (Roth 1991; Shur 1991; Colley 1997).
Two possibilities can be considered to explain the similarity of the phenotypes of EndoGalC transgenic mice and β4GalT-1 KO mice. Firstly, synthesis of the Gal epitope might be inhibited in β4GalT-1 KO mice, and the common phenotype might be caused by the reduction in the level of the Gal epitope. Although such phenotypes have not been reported in 3GalT-1 KO mice (Thall et al. 1995; Tange et al. 1996), one could argue that 3GalT-2 might have compensated for the loss of 3GalT-1. However, it should be kept in mind that 3GalT-2 has so far only been reported to form the Gal1-3Galβ1-4Glc linkage to ceramide and it is not resolved whether EndoGalC acts on this linkage. In general, the degree of contribution of 3GalT-2 to the formation of the Gal epitope has not been established.
A more likely explanation is that the N-acetylglucosamine residue exposed in both EndoGalC transgenic mice and β4GalT-1 KO mice is the origin of the harmful effects. The phenotypes of β4GalT-1 KO mice are mainly ascribed to the lack of completed sugar structures, such as Sia2-3Galβ1-4GlcNAc. The phenotype of EndoGalC transgenic mice thus provides new insight into the phenotype of the KO mice. As described above, the transient skin lesions observed in the young EndoGalC transgenic mice were also observed in the β4GalT-1 KO mice. The skin lesions in the KO mice are thought to be caused by enhanced cellular proliferation due to the altered structure of the cell surface sugar chain (Asano et al. 1997; Lu et al. 1997). However, these lesions disappear 6–10 days after birth (Asano et al. 1997; Lu et al. 1997; this study). Thus, a carbohydrate signal is a critical factor controlling cellular proliferation and differentiation in murine skin during a very restricted stage. A detailed analysis of skin lesions found in young EndoGalC transgenic mice will be reported elsewhere (Misawa et al. 2007 [this issue of Glycobiology]). Our Northern blotting showed that the level of EndoGalC mRNA was higher in the skin of 10-week-old mice than in that of 8-day-old mice (Figure 4A and B). However, only 8–12-day-old mice exhibited skin lesions, which resolved with growth. There appears to be two reasons to explain the apparent discrepancy. Firstly, the mRNA level and protein level of EndoGalC do not always correlate, as mentioned before. Secondly, the substrate level of Gal epitope might be high in the adult mice possibly due to higher expression of glycosyltransferases forming the epitope including β4GalT-1 (Akimoto et al. 1995; Miyagawa et al. 1999).
Although most phenotypic abnormalities manifested in the EndoGalC transgenic mice resemble those in the β4GalT-1 KO mice, there are some discrepancies between the phenotypes of the two types of mouse. For example, the enhancement of cellular proliferation observed in the small intestine epithelium of βGalT-1 KO mice (Asano et al. 1997) was not observed in the EndoGalC transgenic mice. Furthermore, spermatogenesis, fertilizing capacity of sperm, and the formation of the mammary gland were normal in EndoGalC transgenic mice, whereas these processes were abnormal in the βGalT-1 KO mice (Hathaway 2003). These phenotypic alterations observed in β4GalT-1 KO mice (but not in EndoGalC transgenic mice) appeared to be caused by the defect in the sugar chains synthesized by β4GalT-1, such as Sia2-3Galβ1-4GlcNAc-R. Thus, comparison of the phenotypes of β4GalT-1 KO mice with those of EndoGalC transgenic mice may enable dissection of the cause of the abnormality in β4GalT-1 KO mice.
Removal of Gal, the epitope from the organs of livestock animals by genetic engineering has been suggested as a promising approach for xenotransplantation (Sandrin et al. 1997; Ezzelarab and Cooper 2005). Transgenesis based on overexpression of the EndoGalC gene appears to be suitable for that purpose because it can be performed more readily than production of KO mice lacking enzymes (such as 3GalTs) that mediate Gal synthesis (Cooper 1998; Milland et al. 2005). Here, we have shown by flow cytometry and histochemical analyses that the Gal epitope was almost completely removed from the skin, heart, and lung of the young but well grown up animal. Decrease of Gal epitope was not detected by a histochemical method in the kidney, although it was revealed by flow cytometry analysis of cells from the adult organs. Thus, it is likely that the present method is applicable to at least some restricted organs of livestock.
Another potential problem with the generation of EndoGalC transgenic animals is the risk of the development of abnormal phenotypes due to systemic EndoGalC expression. Therefore, employment of tissue-specific promoters conferring limited expression of EndoGalC may be needed. It should also be noted that we used actin promoter to express EndoGalC. This might have resulted in excessive production of the enzyme. Alternatively, introduction of a Cre-loxP-based inducible gene expression system (Rajewsky et al. 1996; Sauer 1998) may be required for controlled expression of the EndoGalC gene beyond the critical stage immediately after birth.
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Protocols for the use of animals in this study were approved by the Animal Care Committee of the National Institute of Agrobiological Sciences.
The vector for production of transgenic mice
For production of EndoGalC transgenic mice, pCAGGS/GT+EndoGalC (Ogawa et al. 2002; Figure 1A) was modified to construct an EndoGalC-expressing plasmid, pCAG-GT-Endo. A fragment containing GT+EndoGalC was first PCR-amplified with primers 5'-GCAGCCGAATTCGATGAGAATGAATATGTACT-3' and 5'’-GCGTCGCTCGAGTTATTTATTTTGTAAAAAAGTTAACTTTAGC-3', and then a signal peptide sequence (102 bp) at the 5' end of GT+EndoGalC was enzymatically removed. A 282-bp fragment containing the transmembrane domain region of the 1,3-GT cDNA, which had been PCR-amplified with primers 5'-CGACGCCTCGAGATGAATGTCAAAGGAAGAGT-3' and 5'-GCGCTGGAATTCAGGATTAAACCAGTCCACTA-3', was ligated to the 5' end of the EndoGalC gene lacking the original trans-membrane domain region. The resulting hybrid fragment was subcloned into the Eco RI sites of pCAGGS, a mammalian expression vector with a strong and ubiquitous chicken β-actin-based promoter (CAG) (Niwa et al. 1991). A 4.98-kb fragment containing GT+EndoGalC was isolated by digestion of pCAGGS/GT+EndoGalC with Sal I and Bam HI, followed by fractionation by electrophoresis through and isolation from a 0.8% agarose gel prior to pronuclear injection of DNA into fertilized eggs.
Transgenic mouse production
The transgene isolated from the pCAGGS/GT+EndoGalC plasmid was microinjected into the pronuclei of fertilized eggs of BDF1 (a hybrid between C57BL/6N and DBA/2; purchased from Charles River Japan, Inc., Tokyo, Japan) mice, according to (Hogan et al. 1994). Transgenic founder (F0) mice were identified by observation, because a skin abnormality appeared soon after delivery (Figure 2D). However, most of these abnormal pups died shortly after birth. Therefore, the surviving F0 transgenics were identified by molecular biological methods (including PCR and genomic Southern hybridization) at weaning. All surviving F0 transgenics were crossed with nontransgenic C57BL/6 mice (aged 8–12 weeks) to obtain F1 offspring. Heterozygous F1 transgenics (aged 8–20 weeks) were subject to Southern blot analysis for identification of transgenes. In some cases, Northern and Western blots were performed to evaluate the expression of EndoGalC mRNA and protein, respectively. These GT+EndoGalC-expressing heterozygous F1 transgenic mice were crossed so as to obtain homozygous transgenic F2 offspring. Mice were kept on a 12 h light/12 h dark schedule (lights on from 0900 to 2100 h) and allowed food and water ad libitum. Experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of National Institute of Agrobiological Sciences. Every effort was made to minimize the number of animals used and their suffering.
Transgene analysis
Genomic DNA was extracted from tissues obtained by biopsy from either live mice or animals that had died shortly after birth, as described by Blin and Stafford (Blin and Stafford 1976). For PCR analysis, 1 µL of DNA (0.05 µg) was dissolved in 24 µL of PCR buffer (10 mM Tris-HCI, pH 9.0, 50 mM KCI, 1.5 µM MgC12 and 1% Triton X-100) containing 0.2 mM dNTPs, 0.2 mM primers, and 50 U/mL Taq polymerase (Applied Biosystems, Foster City, CA). PCR was performed using the following protocol: 96°C for 45 s, 58°C for 25 s, and 72°C for 3 min (30 cycles). Primers used were Forward 1035–1056(5’-tgagtacgttgaaccacaatcc-3'') and Reverse 1731–1711 (5'-accatctccaacttgtgtctc-3'). This primer set yields a 696-bp product; products were analyzed by electrophoresis on a 1.5% agarose gel, and visualized under UV illumination after staining gels with ethidium bromide. In this case, 1 pg of pCAG-GT-Endo vector DNA was used in the PCR reaction as a positive control. For the negative control, 1 µL of genomic DNA (0.05 µg) isolated from C57BL/6 liver was also subjected to PCR. 
phage DNA digested with Hin dIII (#DNA-010; Toyobo, Tokyo, Japan) was used as molecular weight marker.
For genomic Southern blot analysis, genomic DNA (10 µg) was digested with Xho I, thereby producing a 5.8-kb fragment containing the EndoGalC cDNA sequence. After electrophoresis of the enzyme-digested DNA on a 1.0% agarose gel, the DNA was transferred onto Hybond-N+ filters (Amersham Pharmacia Biotech, Little Chalfont, UK). These filters were hybridized with a 32P-labeled Hind III-digested EndoGalC cDNA probe, then exposed to an Imaging Plate (Fuji Film, Tokyo, Japan) overnight, and then analyzed with the aid of a FLA-3000G image analyzer (Fuji Film).
Analysis of viability
Analysis of viability was performed as described by Lu et al. (Lu et al. 1997). Transgenic males were housed with C57BL/6 females. Newborn pups were weighed daily until 15 days after birth. They were also genotyped by PCR by sampling a small piece of the toes at around day 7 after birth. The body weight of the pups was measured every 24 h after the day of delivery.
Northern blot analysis
Total RNA was extracted from mouse tissues using the Isogen RNA extraction reagent (#311-02501; Wako Pure Chemicals Co. Ltd, Tokyo, Japan), following the protocols described by the manufacturers. Using MOPS buffer containing formaldehyde, 10 µg of RNA was separated by electrophoresis through a 1% agarose gel. The RNA was finally transferred onto Hybond-N+ filters. These filters were hybridized to a 32P-labeled EndoGalC cDNA probe, then exposed to an Imaging Plate (Fuji Film), and analyzed with the aid of a FLA-3000G image analyzer.
Generation of anti-Endo-β-galactosidase antibody
Rabbit antisera against TDIQLAKVTFLQNK synthetic peptides corresponding to the C-terminus of EndoGalC was raised and subsequently affinity-purified using Sulfo-SMCC (#22322; Pierce Chemical Co., Rockford, IL) according to the protocol described by the manufacturer. The purified polyclonal antibody was termed AE-1. Specificity of AE-1 for EndGalC was verified by blocking experiments with the peptides used for the immunizations.
Western blot analysis
Dissected organs were homogenized in a sample buffer [10% sucrose (w/v), 3% SDS (w/v), 60 mM Tris-HCl (pH 6.8)] and then the supernatants were treated with β-mercaptoethanol. They were separated by electrophoresis under reducing conditions through an 8% polyacrylamide-SDS-gel and transferred to nylon membranes (Immobilon-P; Millipore, Bedford, MA). The membranes were next blocked with 5% nonfat dry milk in Tris-buffered saline [TBS; 50 mM Tris-HCl (pH 7.4), 150 mM NaCl], and then incubated with AE-1 at the concentration of 2 µg/mL. After washing with TBS, the membranes were incubated with horse radish peroxidase (HRP)-linked anti-rabbit IgG (#NA934VS; Amersham) diluted 5000-fold in TBS containing nonfat dry milk. The membranes were then rewashed with TBS. The EndoGalC proteins were detected by treatment of the membranes with the ECL Plus Western blotting reagent (#RPN2132; Amersham) and subsequent exposure to an X-ray film for several minutes at room temperature. The membranes were washed with WB stripping solution (#05364-55; Nacalai Tesque Co., Kyoto, Japan) to remove antigens after exposure to X-ray film and blocked again in 5% nonfat milk containing TBS.
Flow cytometry analysis of EndoGalC expression in fibroblast cells derived from transgenic mice or embryos
The presence or absence of the Gal epitope on the surface of MEFs derived from EndoGalC transgenic mice was determined using a flow cytometer, as described by Ogawa et al. (Ogawa et al. 2002). Pregnant female mice were sacrificed at Day 14 of gestation (Day 0 is defined as the day when a copulation plug was first observed) and fetuses were dissected. The associated yolk sac and amnion were removed from the fetuses under germ-free conditions. After washing with Dulbecco's modified phosphate-buffered saline (PBS) without Ca2+ and Mg2+ [PBS(–)], fetuses were minced using scissors, and dispersed by incubating them at 37°C for 20 min in 10 mL of PBS(–) containing 0.1%(w/v) of collagenase (#C9407; Sigma-Aldrich, St Louis, MO) and 0.1%(w/v) of trypsin (#159090-46; Gibco, Grand Island, NY). Dissociated cells were then collected by low-speed centrifugation at 4°C and then resuspended in PBS(–). The number of viable cells was counted using the improved Neubauer hemocytometer (Kayagaki, Tokyo, Japan) after staining with trypan blue. Obtained cells were seeded and cultured in a 100-mm plastic dish (#3003; Becton Dickinson, Franklin Lakes, NJ) with 10 mL of Dulbecco's modified Eagle's medium (DMEM) (#D5796; Sigma-Aldrich) containing 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 in air until confluent. The cells were trypsinized and used for the analysis. Cells isolated from adult organs (i.e. heart, lung, kidney, and pancreas) dissected from the 6-month-old animals were treated similarly. The cells from adult animals were analyzed immediately after isolation to prevent possible phenotypic alteration during culture of isolated cells.
The dispersed cells were collected and then stained with FITC-labeled GS-IB4 isolectin (#L2895; Sigma-Aldrich) at a concentration of 20 µg/mL on ice for 30 min in PBS/BSA [PBS(–) containing 0.2% (w/v) bovine serum albumin and 0.1% (w/v) sodium azide]. After incubation on ice, the cells were washed twice with PBS/BSA and then the stained cells were suspended in 0.5 mL of PBS/BSA, and analyzed using flow cytometry (Epics XL-MCL; Beckman Caulter, Fullerton, CA). The mean fluorescence intensity (MFI) was used to quantify the expression of Gal.
Hematoxylin/eosin staining
For hematoxylin/eosin (HE) staining, transgenic and nontransgenic skin samples were fixed in 4% paraformaldehyde embedded in paraffin and sectioned at 7-µm thickness. The slides were deparaffinized in xylene and hydrated gradually through an alcohol gradient. After staining for 2 min in hematoxylin, the sections were incubated for 5 min in water. They were next stained in eosin for 3 min and briefly washed in water. Finally, samples were dehydrated and mounted. Photographs were taken using an Olympus BX50 microscope (Olympus, Tokyo, Japan).
Immunohistochemistry and lectin staining of mouse tissues
For immunohistochemistry, paraffin sections were deparaffinized, hydrated, and incubated in citric acid buffer [0.2% (w/v) citric acid, pH 6.0] at 95°C for 40 min, washed in PBS (–), and blocked for 30 min at room temperature in PBS(–) containing 1% (w/v) BSA. They were then incubated with AE-1 (diluted 1:300) antibody overnight at 4°C. After washing with PBS(–), the sections were next reacted with Alexa Fluor 594 donkey anti-rabbit IgG (diluted 1:2000) for 30 min at room temperature. Similarly, sections were incubated in 3 µg/mL solution of GS-IB4 isolectin conjugated with FITC (Sigma-Aldrich) at room temperature, and then washed with PBS(–) for detection of the Gal epitope. Sections were also incubated in 20 µg/mL of GS-II isolectin conjugated with Alexa Fluor 488 (derived from Griffonia simplicifolia; L21415
[GenBank]
; Molecular Probe Co., Carlsbad, CA) for detection of the GlcNAc residue exposed at the terminus of the sugar chain. The sections were finally reacted with 0.5 µg/mL of DAPI (#09224; Polysciences, Inc., Warrington, PA) for counter staining of the nuclei. Sections were mounted in Perma Fluor mount medium (Thermo Shandon, Pittsburgh, PA). Fluorescence in the sections was observed using an Olympus BX50 fluorescence microscope (Olympus) with DM505 filters (BP470-495 and BA510-550IF) and DM600 filters (BP545-580 and BA6101F), which were used for Alexa Fluor 594 and FITC monitoring, respectively. Microphotographs were taken using a chilled monochrome change-coupled (CCD) camera (Penguin 150CLM; Pixera, Kanazawa, Japan) attached to the fluorescence microscope and substituted with pseudo-colors.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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Ministry of Education, Science, Sports, Culture and Technology of Japan (17100007).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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None Declared.
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Acknowledgements |
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The authors would like to thank Mrs. Sachiko Watanabe for technical assistance with the molecular biological analysis and animal care. The authors also would like to thank Dr. Hirohide Uenishi for his technical assistance with the flow cytometry analysis.
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Abbreviations |
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β4GalT-1, β1,4 galactosyltransferase 1; EndoGalC, Endo-β-galactosidase C; FITC, fluorescein isothiocyanate; HRP, horse radish peroxidase; KO, knockout; MEF, embryonic fibroblast cells
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