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TOPICAL REVIEW |
1 Molecular Physiology Laboratory, University of Edinburgh Medical School, Edinburgh EH8 9AG, UK2 Roslin Institute, Roslin, Midlothian EH25 9PS, UK
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Abstract |
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(Received 23 June 2003;
accepted after revision 16 October 2003;
first published online 17 October 2003)
Corresponding author J. J. Mullins: Molecular Physiology Laboratory, University of Edinburgh Medical School, Edinburgh EH8 9AG, UK. Email: j.mullins{at}ed.ac.uk
Factors contributing to cloning inefficiency
Enucleation and subsequent nuclear transfer of a diploid nucleus into the oocyte places enormous demands on the cell. Firstly, the oocyte has to survive the physical, or chemical removal of its own haploid nucleus, endure in vitro culture conditions, and then survive further physical insult through replacement of nuclear material (whether by microinjection or electro-fusion). Needless to say, there are significant losses at each stage of the nuclear transfer process. The timing of oocyte activation relative to nuclear transfer must be taken into consideration (Wakayama et al. 2000). The oocyte cytoplasm must then reprogramme the incoming nucleus, such that patterns of gene expression will mimic, as closely as possible, those which would normally occur following fertilization by the sperm, and during blastomere formation. Time constraints dictate that the reprogramming should be complete before cell-fate decisions are made regarding trophoblast differentiation, implantation and the development of extra-embryonic and embryonic tissues.
Trophoblast cells give rise to much of the placenta and some extraembryonic membranes. Thus, trophoblast malfunction, leading to placental insufficiency, is a significant complication of somatic cell nuclear transfer, leading to postimplantation developmental arrest at fetal, and perinatal stages. Typically, over one-third of cloned embryo pregnancies in cattle and sheep abort during gestation (Paterson, 2002). In cattle, the late-gestation losses are particularly striking, with up to 40% of somatic nuclear transfer pregnancies aborting post Day 90 of gestation, compared with 4.3% after embryo cloning (fusion of blastomeres to enucleated oocytes) and 0% following in vitro fertilization (Heyman et al. 2002). The overaccumulation of placental fluid in hydroallantois (which occurs rarely in natural pregnancies) can affect up to 40% of cloned embryo pregnancies. Though the placentation processes in cattle and mice are quite different, both placentomegaly and increased birthweight have also been reported in mouse cloning, regardless of the source of donor nuclei (Wakayama & Yanagimachi, 1999; Ogura et al. 2000; Eggan et al. 2001). In cloned mice, the placentae are two- to threefold larger than those of normal non-cloned fetuses, and show an expansion of the spongiotrophoblast layer, which disturbs placental architecture and damages its function (Tanaka et al. 2001).
Further losses due to neonatal deaths, and the production of abnormal offspring are also common following nuclear transfer. In cattle, the mean birth weight of adult somatic cloned calves was significantly higher than that of IVF calves, and the survival rate was lower (Heyman et al. 2002). Many cloned offspring die within the first 24 h of birth, often because of respiratory distress or cardiovascular dysfunction. Further complications include prolonged gestation, fluid accumulation, and enlarged organs. Some of these complications may not be specific to nuclear transfer, but their causes must be addressed if nuclear transfer is to be commercially viable, and efficiency is to be improved.
Some phenotypic effects may result from the donor cell type. Cloned mice produced by microinjection of adult cumulus cell nuclei into enucleated oocytes grew to be obese (Tamashiro et al. 2002). The obesity, however, was eliminated or ‘corrected’ during gametogenesis, since the obesity was not passed on to progeny. This suggests that the phenotype might be due to epigenetic modification rather than a genetic change. In another example, using immature Sertoli cells as nuclear donors, cloned mice were found to die at an unusually early age (Ogonuki et al. 2002; Ogura et al. 2002).
Nuclear transfer and oocyte activation
Enucleation of oocytes is achieved mechanically, using micromanipulation. The donor nucleus can be introduced into the oocyte by microinjection or fusion. The transferred nucleus undergoes disassembly in response to high levels of maturation promoting factor (MPF) in the metaphase II (MII) cytoplasm. (When oocytes enucleated at the pro-metaphase I stage and cultured to the MII stage were compared with oocytes enucleated at metaphase II, the proportion of reconstituted embryos which developed to the blastocyst stage were 8.1% and 53.5%, respectively (Gao et al. 2002), suggesting that the more mature oocyte is a better recipient for nuclear transfer.)
Following artificial activation (either by chemical or electrical treatment) of the reconstituted oocyte, nuclear reassembly occurs. A number of groups have addressed the developmental effect of timing of oocyte activation relative to nuclear transfer. Wakayama et al. (2000) injected somatic cell nuclei into enucleated, unfertilized oocytes, using piezo-electrically driven micromanipulation. The oocytes were activated (with strontium chloride) 1 h before (preactivation), immediately after (immediate activation) or 1–3 h after nuclear transfer (postactivation). In vitro development to the blastocyst stage was scored at 6%, 34% and 69%, respectively, suggesting that activation following nuclear transfer may be beneficial but is not essential. (The same is true in cattle (Kubota et al. 2000), but not in sheep, where late activation has no effect.) Reconstituted oocytes, which were activated immediately after or 1–3 h after nuclear transfer produced live offspring at similar frequencies (see Table 1) (Wakayama & Yanagimachi, 2001a). Neither activated zygotes nor activated oocytes were appropriate recipients for nuclear transfer.
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Clearly, it is necessary to confirm nuclear contribution in nuclear transfer experiments. Such contribution can be easily demonstrated in progeny, if different donor and recipient coat colours are involved, for example. However, the contribution in nuclear transfer embryos is more difficult to demonstrate. To visualize nuclear contribution, nuclear localized lacZ expression (Zin40) transferred with the donor nucleus, is a useful tool (Munsie et al. 1998).
Somatic cell nuclear transfer
The first nuclear transfer experiment involved nuclei from an embryo-derived cell line, which had been induced to become quiescent (Campbell et al. 1996). It was soon demonstrated that viable offspring could be derived from quiescent embryo-, fetal- and adult-derived mammalian cells (Wilmut et al. 1997). This striking result suggested that even adult differentiated cells were not irreversibly genetically modified, and their DNA could be reprogrammed to direct embryo development. Since then, a large number of different donor somatic cell types have been used successfully in nuclear transfer.
Nuclei from two-, four- and eight-cell embryos taken at the early stage of each cell cycle, were injected into enucleated oocytes, and following electrofusion, chromosome condensation was observed. At this point, further electrical stimulation was applied to activate the embryos. These developed into blastocysts, and subsequently a high proportion (see Table 1) developed to term (Cheong et al. 1993). Four-cell embryo nuclei, arrested at metaphase, were successfully used to generate cloned mice, by a serial nuclear transfer technique (Kwon & Kono, 1996). The donor embryos were cultured with nocodazole (a microtubule polymerization inhibitor) to induce metaphase arrest. Each nucleus was introduced with inactivated Sendai virus into the perivitelline space of an enucleated oocyte, and after artificial activation by electrical stimulation, the reconstituted egg was cultured in the presense of cytochalasin B, to inhibit polar body extrusion and induce the formation of two ‘pronuclei’. The latter were transferred to enucleated one-cell zygotes and 83% developed into blastocysts. On transfer to recipient mice, 57% developed to term. It is suggested that the serial transfer allowed more time for reprogramming of the metaphase nucleus to take place. These results are significant because in mice, reprogramming of embryonic nuclei appears to be restricted after embryonic genome activation, which occurs at the two-cell stage.
Sertoli cells and neuronal cells (both non-dividing cells), and cumulus cells (90% of which are in the G0/G1 phase of the cell cycle when harvested with superovulated oocytes) were chosen to investigate the development potential of oocytes injected with nuclei from non-cultured cells (Wakayama et al. 1998). Oocytes receiving adult Sertoli or neuronal nuclei developed in vitro, and implanted following transfer, but did not develop beyond 8.5 days post coitum. A high percentage of oocytes receiving cumulus cell nuclei were transplanted, and though most were resorbed, some developed to term (see Table 1). More recently, cloned females derived from adult cumulus cell nuclei were found to exhibit an obese phenotype, which was not passed on to progeny (Tamashiro et al. 2002). It was suggested that the phenotype could result from nuclear transfer or in vitro culture conditions, but in vitro embryo-manipulated controls failed to show the obese phenotype, which discounts embryo manipulation, culture, transfer into pseudopregnant females, reduced litter size, Caesariean section delivery and cross-fostering as contributing factors.
The first male-derived clones were generated using donor cells cultured from adult male tail-tips and three of the transferred embryos reached full-term (see Table 1) (Wakayama & Yanagimachi, 1999). Fetal fibroblast cells, arrested at metaphase, resulted in severely abnormal stillborn embryos, following a single nuclear transfer protocol, but were successfully used to generate cloned mice, by serial nuclear transfer (see Table 1) (Ono et al. 2001). Immature Sertoli cells, isolated from the testes of newborn mice (days 3–10) were found to produce viable offspring following nuclear transfer. This ability was maintained, after 1 week in culture, and even after cryopreservation (Ogura et al. 2000). Recently, it has been reported that the lifespan of mice cloned from immature Sertoli cells is significantly shorter than that of genotype- and sex-matched controls. This finding suggests the possibility of long-term deleterious effects of somatic-cell cloning (Ogonuki et al. 2002).
Of oocytes reconstituted with neural cells from the cerebral cortex of developing fetuses (15.5–17.5 days pc), 5.5% developed into normal offspring (Yamazaki et al. 2001). This was significantly higher than neural cell nuclei from other areas of the brain, suggesting that nuclei of cells in advanced stages of differentiation had lost their developmental totipotency, perhaps through somatic DNA rearrangements.
Due to the low success rate of somatic cell nuclear transfer, there is formally a possibility that surviving clones are in fact derived from the nuclei of rare stem cells present in adult tissues, rather than from the nuclei of differentiated cells. Evidence that terminally differentiated cells can be reprogrammed to produce adult cloned animals has come with nuclear transfer from mature B and T donor cells (Hochedlinger & Jaenisch, 2002). During lymphocyte maturation in bone marrow and thymus, B and T cells undergo genomic rearrangements at the immunoglobulin and T cell receptor loci, respectively. Using peripheral lymphocytes as donors, direct nuclear transfer of cloned blastocysts was unsuccessful (Wakayama & Yanagimachi, 2001b). However, using a two-step procedure, ES cells were derived from the cloned blastocysts, followed by tetraploid embryo complementation (Nagy et al. 1993). By this strategy, the placenta is derived from tetraploid embryo cells, and the embryo from the injected donor ES cells. The overall efficiency of the procedure was low, but two ES cell lines exhibiting the genomic rearrangements were isolated and viable progeny arising from one of these were generated by tetraploid complementation (Hochedlinger & Jaenisch, 2002). All animals cloned from the B-cell nucleus carried fully rearranged immunoglobulin genes in all tissues. The authors suggest that terminally differentiated lymphocyte nuclei may only rarely be reprogrammed on transfer into the oocyte. The ES cell intermediate stage may allow more time for faithful reactivation of embryonic genes.
ES cell nuclear transfer
ES cell lines are derived from the inner cell mass of embryos at the blastocyst stage, and can be cultured in vitro for many passages without becoming aneuploid. Importantly, they exhibit developmental pluripotency and can be used to generate chimeric mice on injection into host blastocysts, either diploid (Hooper et al. 1987) or tetraploid (Nagy et al. 1993). The ES cells then contribute to all embryonic tissue in the developing fetus.
ES cells have also been found to be capable of directing the clonal development of viable offspring (Wakayama et al. 1999). Because rapidly dividing cell populations are asynchronous, the choice of nucleus donor was made according to cell size. Small cells were surmised to be in G1-phase (2C) while large cells corresponded to those in G2/M-phase (4C). Following injection into enucleated metaphase II oocytes, the cells were strontium-activated in the presence or absence of cytochalasin B, respectively, to ensure correct ploidy of the resultant embryo. Two different ES cell lines were used, and live offspring were generated from both, though only mice derived from the R1 ES cell line survived in any numbers. Notably, these pups were produced from nuclei at both G1- and G2/M-phase. A separate study found that significantly higher numbers of metaphase II nuclei developed to the blastocyst stage than interphasic nuclei (see Table 2) (Zhou et al. 2001). Though the implantation rate of one- or two-cell embryos was high (32%), they noted high levels of mortality post-implantation.
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It was initially thought that only ES cells derived from intercross (and hence heterozygous) animals were effective nuclear donors, but mice were recently cloned from the inbred ES cell line, HM-1, derived from 129/Ola (Gao et al. 2003). Further characterization of ES cell karyoplast function determined that ES cell confluence dramatically affected the developmental potential of reconstituted embryos. At 80% confluence, using the inbred HM-1 ES cells as nuclei donor, nearly a third of live pups survived to adulthood (see Table 2). At 60–70% confluence, only one live pup was born (Gao et al. 2003). Previously, it was noted that cell passage number dramatically affected the ability of ES cells to aggregate with tetraploid embryos, and give rise to viable progeny (Nagy et al. 1993). A similar decrease in cloning efficiency with passage number has been reported (see Table 2) (Gao et al. 2003). Using the intercross-derived ES cell line, R1, at passage 19, three live pups were born and one reached adulthood. At passages 22–25, no fetuses survived.
Additional determinants of nuclear transfer
Telomere length
The relationship between donor age and proliferative lifespan is pertinent to the discussion here. Normal diploid fibroblasts have a finite replicative lifespan in vitro, which is postulated to be a cellular manifestation of ageing in vivo. In cattle, it was reported that fibroblasts derived from cloned fetuses grew significantly longer in culture than those from age-matched control fetuses (Lanza et al. 2000). No evidence for extension of proliferative lifespan could be found in a study on sheep fetal fibroblasts (Clark et al. 2003). However, nuclear transfer was shown to reprogramme cellular lifespan to that of the donor cell line. That is to say, the cell line from which the donor nucleus originates strongly determines the proliferative capacity of cell lines rederived after nuclear transfer.
A major control of proliferative lifespan of cells is the telomere. Vertebrate telomeres are long tandem arrays of hexameric DNA sequences (TTAGGG)n located at the ends of chromosomes, which regulate stability, replication and segregation of chromosomes during division. Telomeres get progressively shorter with each round of cell division, until they reach a critical length. At this point they lose the capacity to effectively cap chromosomes, and DNA damage response pathways then lead to cell cycle arrest or senescence. A number of groups have assessed telomere length before and after nuclear cloning. In cattle, telomere lengths were not significantly different (15–23 kb) in cloned fetuses compared with age-matched controls, but were longer than in the adult donor cells (14–18 kb). This suggests that cloned embryos inherit genomic modifications acquired throughout the in vivo and in vitro development of the donor nucleus, but that they are reversed during development of the cloned animal (Betts et al. 2001). Similar observations were made in sheep (Clark et al. 2003). It has been reported that Dolly, had telomeres that were significantly shorter than those of age-matched controls at one year of age (Shiels et al. 1999; Shiels & Jardine, 2003). Dolly was cloned from a mammary cell line derived from a six-year-old ewe. This suggests that nuclear transfer may extend telomere length, but not necessarily restore it to full length. It should be noted that ultra-long telomeres have been observed in mice, which would not apparently agree with the concept that critical telomere shortening is related to ageing. Careful analysis of telomere lengths in various mouse strains indicated that the mouse does indeed have some short telomeres (10 kb) and these may be critical in limiting the replicative potential of murine cells (Zijlmans et al. 1997).
Imprinting
Early passage ES cells can be used to produce completely ES cell-derived fetuses (Nagy et al. 1990). However, upon prolonged culturing the potential of many ES cell lines becomes impaired, resulting in ES-derived fetuses with abnormalities. High passage stem cells may even cause abnormalities in chimeras, resulting in postnatal death (Nagy et al. 1993). Loss of developmental potential appears to result from the accumulation of epigenetic alterations in DNA methylation. During preimplantation development, the bulk of DNA in the genome becomes unmethylated, so that by the blastocyst stage, when ES cells are derived, levels of methylation are low. Fetal levels of methylation are acquired after implantation, by de novo methylation (Monk, 1987; Kafri et al. 1992). For imprinted genes, whose expression is parent-of-origin dependent (for gene dosage control), regulation of methylation is somewhat different. Imprinted genes show parental allele-specific methylation (John & Surani, 1996), and have considerable levels of methylation at the blastocyst stage. The differentially methylated regions (DMRs) marking the parental allele to be either expressed or silenced (depending on the gene), appear to be established or reinforced during gametogenesis and early embryogenesis (Latham, 1999). It is therefore plausible that in vitro embryo culture or manipulation at the time when imprints are established might be a mechanism for induction of imprinting errors. It was found that alterations in imprinted gene methylation and expression arising during the culture of ES cells persisted in ES-derived mouse fetuses and was associated with aberrant development (Dean et al. 1998). It was therefore suggested that improper expression of imprinted genes might also contribute to the abnormalities observed in cloned offspring (Young & Fairburn, 2000).
Epigenetic changes were investigated in large offspring (LO) following sheep embryo culture, when it was found that expression of IGF2R was reduced by 30–60% in large fetuses compared to controls. Restriction analysis of the second intron DMR revealed a complete loss of methylation in 9 out of 12 LO individuals compared to 70% methylation of the CpG island in controls (Young et al. 2001).
Since a significantly higher fraction of blastocysts cloned from ES cell nuclei survive to adulthood than any somatic cell type (Rideout et al. 2000; Eggan et al. 2001), it was surmised that the nucleus from an undifferentiated cell is more amenable to reprogramming. Imprinted gene expression was therefore analysed in cloned mice, and the ES-cell donor populations from which they were derived. Expression and methylation of imprinted genes varied widely in the placentas and tissues of cloned animals, but in a given placenta, abnormal expression of one imprinted gene did not correlate with abnormal expression of another (Humpherys et al. 2001). To investigate whether faulty programming accounted for the observations, imprinting in the donor cell population was investigated. Again, expression of imprinted genes varied widely between individual ES-cell subclones. Surprisingly, expression was significantly different among pups cloned from the same ES subclone, suggesting that the epigenetic state of ES cells is very unstable (Humpherys et al. 2001).
Studies on the expression of imprinted genes following somatic cell nuclear transfer indicated that the choice of parental allele expressed reflected that described for fertilization-derived embryos, in all fetuses and placentas surveyed, and for all imprinted genes investigated (Inoue et al. 2002). This suggested that the imprinting memory established during gametogenesis is stable, and not readily transmuted within oocytes or embryos. Steady state mRNA levels for both imprinted and non-imprinted genes fell within the control range in fetal and neonate tissues (though placental levels were reduced). Transcript levels for Ig f2 and H19, which are prone to anomalous expression in ES-derived nuclear transfer, were also found to lie within normal range (Inoue et al. 2002).
To assess the global extent of abnormal gene expression in clones, microarray analysis on RNA from the placentas and livers of neonatal cloned mice derived from both cultured ES cells and freshly isolated cumulus cells was assessed (Humpherys et al. 2002). Direct comparison of more than 10 000 gene expression profiles revealed that 4% of expressed genes in the NT placentas differed dramatically from control levels. The majority of these were common to both types of clones. However, a small set of genes differed between the embryonic stem cell-derived and cumulus cell-derived clones. The livers of cloned mice showed abnormal gene expression to a lesser extent, and in a different subset of genes from the placenta. Interestingly, H19 reduction was specific to ES-cell-derived animals, and may reflect sensitivity to environmental influences such as in vitro cultivation. Similar analysis of animals derived from ES cells by tetraploid complementation should allow one to distinguish genes affected specifically by nuclear transfer from those affected by characteristics of the ES cell donor.
To determine when imprinting occurs, oocytes at different stages of growth were introduced into metaphase II-arrested oocytes. The oocytes were fertilized in vitro, and blastocysts were transferred to recipients to assess development and expression of a number of paternally expressed and maternally expressed genes. Results showed that the genes were not all imprinted at the same time, but rather epigenetic modifications for each imprinted gene occurred independently, in a specific sequence throughout the period from primary to antral follicle stage oocytes (Obata et al. 1998; Bao et al. 2000; Obata & Kono, 2002).
Application of nuclear transfer to the rat
The vast body of research into nuclear transfer has been carried out in the mouse. Since the rat has classically been the species of choice for cardiovascular, pharmacological and neurological research, it would be highly advantageous to apply the full range of molecular and genetic modifications to this species. In 1982, it was reported that rat oocytes spontaneously activate during in vitro culture, and this was affected by changes in oviduct conditions during oocyte removal (Keefer & Schuetz, 1982). This potential problem may be reduced by the removal of oviducts into calcium-free PBS (Hayes et al. 2001).
In 1988, a reliable method for superovulation of rats was published (Armstrong & Opavsky, 1988). This was swiftly followed by the development of transgenesis by microinjection, allowing gene over-expression and induction (Mullins et al. 1990; Kantachuvesiri et al. 2001). To date, ES cell technology in the rat has proved to be intractable, disallowing the possibility of gene knockout technology. Differences have been observed in the differentiation potential of epiblast tissue from mouse and rat embryos (Nichols et al. 1998), the latter showing a propensity for producing parietal endoderm. ES cell-like cells have been reported (Vassilieva et al. 2000), which express alkaline phosphatase and SSEA-1 (the latter somewhat patchily). Similar cells were shown to differentiate into haematopoietic cells on introduction into adult rats (Fandrich et al. 2002). It has been found that Oct-4, a POU transcription factor, which is essential for the pluripotent character of the mouse inner cell mass and derivative ES cells, is completely down-regulated in cultures from rat blastocysts and ICMs. This appears to be a limiting factor in attempts to derive ES cell lines from preimplantation embryos (Buehr et al. 2003). Although blastocyst-derived cells do not retain pluripotency, they have been cultured to high passage numbers (>50) and are transfectable. They could therefore be used for targeted genetic modification, and the nuclei could be transferred to recipient oocytes to generate transgenic rats. It will be interesting to see if the recently published homeoprotein, Nanog, which is also critical for pluripotency in mouse ICM and ES cells (Cavaleri & Scholer, 2003; Chambers et al. 2003; Mitsui et al. 2003), is found to be limiting in rat ES cell generation. Knock-out of Nanog in mouse ICM leads to the production of parietal endoderm, and in ES cells, leads to their differentiation into extraembryonic endoderm lineages (Mitsui et al. 2003). Overexpression of Nanog transgene constructs, on the other hand, is sufficient for clonal expansion of ES cells (Chambers et al. 2003).
Recently, gene knockout in the rat has been achieved using an ENU mutagenesis protocol, combined with a yeast-based screening assay to identify functional mutations in the target gene of interest (Zan et al. 2003). ENU was administered at a level which generated one visible phenotypic mutation per 64 pups. Mutants in the two target genes were identified after screening 788 and 2000 pups, respectively. Whilst this is not a readily available technique, it circumvents the requirement for ES cells to knock-out any gene of interest. Given the lack of ES-cell technology, it would be highly desirable to achieve nuclear transfer in the rat, but until recently attempts have proved only partly successful.
Historically, the rat embryo was refractory to in vitro culture, exhibiting a complete developmental block at the two- to four-cell stage in modified Krebs–Ringer bicarbonate medium (mKRB) following in vitro fertilization (Toyoda & Chang, 1974). Modifications to the culture medium have overcome this block (Miyoshi et al. 1997), and improved in vitro fertilization and blastocyst development to the point where live births have been reported (Oh et al. 1998).
Nuclear transfer was attempted in the rat using a zygote as both donor nucleus and recipient cytoplasm, and live births were reported (Kono et al. 1988). More recently, embryonic fibroblast nuclei (Fitchev et al. 1999), and adult and genetically modified (transfected) rat fetal cells (Hayes et al. 2001), were transferred into rat MII oocytes using a variety of micromanipulation and activation procedures but in neither study were live births reported. Earlier this year, live births were obtained following the transfer of nuclei from two-cell rat embryos into enucleated two-cell embryos, and also using zygote to zygote transfer (Roh et al. 2003). Three of the pups born from these transfers survived, and though variation in fur density was noted, it is not clear if this resulted from the nuclear transfer procedure. Recently, the problem of spontaneous oocyte activation, triggered by inactivation of MPF, has been addressed (Zhou et al. 2003). Collection of oocytes in the presence of a protease inhibitor (MG132) stabilized most oocytes for up to 3 h, allowing time for a successful nuclear transfer. Genetically fertile animals were obtained.
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