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State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cloned fish, common carp, cross-genus, cytoplasmic impact, developmental biology, early development, embryo, goldfish
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Just as in other vertebrates, fish bone skeletal system is made up of repeating patterns, among which the most obvious are the vertebrae. Vertebral patterning is a result of somite patterning during embryogenesis, and the vertebral number varies a lot among different fishes but is relatively stable within a given species [16]. For example, the vertebral number of common carp is 33–36 and that of goldfish is 26–28 [17]. Thus, the vertebral number has been considered an important element in taxonomic study. Nevertheless, we still lack a comprehensive understanding of the molecular mechanism of action that controls the somite number and vertebral number. Nuclear transplantation between two species with different vertebral numbers may provide novel insights into the mechanisms underlying this process.
In the present study, we conducted experiments of cross-genus nuclear transplantation between two fish species with different vertebral numbers, Cyprinus and Carassius [17, 18]. We tested whether the enucleated eggs of goldfish could adapt and reprogram common carp nuclei to direct the embryogenesis and ontogenesis of resulted nuclear transplants. We extensively analyzed the origination of the nuclear and mitochondrial genomes in the NT animals.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The founder of transgenic red common carp, with a long body in shape and two barbels on each side of the mouth, was produced by introducing the recombinant construct MThGH, composed of the human growth hormone gene (hGH) driven by a mouse metallothionein-1 gene promoter (MT), into fertilized eggs via microinjection [19]. The subsequent F1-F3 generations were produced as previously reported [20]. The red-dragon-eye strain of goldfish, which has protruding eyes, triangle tail, and spherical body in shape, was used in this experiment. Use of these animals for experimental purposes was approved by the Scientific Committee at the Institute of Hydrobiology, Chinese Academy of Sciences.
Preparation of Donor Nuclei
Fertilized eggs from transgenic F3 red common carp were cultured in Holtfreter solution (0.35% NaCl, 0.01% CaCl2, 0.005% KCl [w/v], 50 IU/ ml streptomycin, and 100 IU/ml ampicillin) up to the blastula stage. The blastoderms were cut from the yolk with a fine glass needle and placed into Holtfreter dissociation solution (Ca2+-free Holtfreter solution with 0.15 mM EDTA). After 2 min, cells of the blastoderms were dissociated and would be used as donors for nuclear transplantation.
Preparation of Recipient Enucleated Eggs
Goldfish were artificially induced to spawn for egg collection. The unfertilized eggs were placed into a trypsin solution of 0.25% (w/v; Sigma, St. Louis, MO) for 3 min. The softened chorion was subsequently removed by microsurgery. The second polar body of the dechorionated egg was visible under a 40x stereomicroscope. The egg nucleus underneath the second polar body was removed by picking with a sharp glass needle. Enucleated eggs were held in an agar plate filled with Holtfreter solution for further manipulation. Successful enucleation was proved in parallel experiments with Hoechst 33342 (Sigma) staining and examination under an ultraviolet light using Olympus SZX-12 microscope (Japan).
Nuclear Transfer
Using a micromanipulator designed in our lab, a single donor cell was ruptured by gently sucking into a micropipette and then was microinjected into the animal pole of an enucleated egg. The manipulated eggs were carefully placed into Holtfreter solution for development at 19°C, and subsequent development was followed by microscopy. Deformed embryos were removed periodically. The fry that appeared to be developing normally were raised with great care in a glass tank and then in a small pond. For controls, sexual hybrid fish were produced by artificial mating of red common carp males with goldfish females.
Total DNA Extraction
Total DNAs from embryos and fry was prepared as follows: each sample was centrifuged onto the bottom of a microfuge tube and digested with 1 µg each of proteinase K (Sigma) and RNase A (Sigma) in 100 µl of DNA extraction buffer (10 mM Tris·Cl, pH 8.0, 300 mM NaCl, 10 mM EDTA, 2.0% [w/v] SDS). DNA was recovered by serial extractions with phenol and chloroform, precipitated with ethanol, and finally dissolved in 20 µl of TE buffer (10 mM Tris·Cl, pH 8.0, 1 mM EDTA). Total DNAs of the cloned fish were extracted from the tailfin as described [19].
Polymerase Chain Reaction Analysis
Two primers (forward: 5'-GGTAAGCGCCCCTAAAATCC-3' and reverse: 5'-TTGAAGATCTGCCCAGTCCG-3') for detection of hGH-transgene are both located in the hGH coding sequence. The expected amplification size of polymerase chain reaction (PCR) product was 712 base pairs (bp). PCR was performed by using 0.5 U of Taq DNA polymerase (BioAsia, Shanghai, China), 10 pmol of each primer, and 50 ng of total fish DNA (for embryonic and fry samples, 5 µl DNA solution instead) as template in a volume of 25 µl. The reaction process was 94°C for 4 min, 30 cycles of 94°C, 30 sec; 58°C, 30 sec; and 72°C, 1 min. All PCR products were separated by electrophoresis on 0.8% (w/v) agarose gels and visualized using a UVP GDS8000 system.
Random Amplification of Polymorphic DNA Analysis
We used a comparative random amplification of polymorphic DNA (RAPD) analysis to distinguish genomic DNAs of common carp and goldfish. Specific primers that could give unique patterns to common carp or goldfish were selected from 20 random primers (S121–S140; Sangon, Shanghai, China). RAPD reaction mixture contained 1 U of Taq DNA polymerase (BioAsia), 10 pmol of oligonucleotide primer, and 60 ng of total DNA as template in a volume of 20 µl. The process of reaction included 94°C for 4 min; 40 cycles of 94°C, 45 sec; 36°C, 1 min; and 72°C, 1 min. RAPD products were separated by electrophoresis on 1.5% (w/v) agarose gels for visualization.
Amplification of mtDNA
The full sequences of mtDNAs for both common carp and goldfish were downloaded from GenBank (Accession no. NC_001606 and NC_ 002079, respectively). Based on the DNA alignment with DNATools software (5.1 version, S.W. Rusmussen), PCR primers for distinguishing mtDNA from common carp and goldfish were designed. For common carp, the forward primer was 5'-GGAGGTAGCACTCCC-3' (5'-3' position: 1–15) and the reverse primer was 5'-GGGGTTTGTCGCGCA-3' (5'-3' position: 688–702). Both primers are located in the D-loop region of common carp mtDNA and the expected length of the PCR product was 702 bp. For goldfish, the forward primer was 5'-CCTGGCTGCCGGTAT-3' (5'-3' position: 7002–7106) and the reverse primer was 5'-CGTGGTATTCCTGCT-3' (5'-3' position: 7698–7712). Both primers are located in the goldfish cytochrome c oxidase subunit I gene and the expected length of PCR product was 711 bp. The PCR amplifications were conducted under the following parameters: 0.5 U of Taq DNA polymerase (BioAsia), 10 pmol of each primer, and 100 ng of total DNA (for embryonic and fry samples, 5 µl of DNA solution instead) in a total volume of 25 µl; 94°C, 4 min, 30 cycles of 94°C, 30 sec; 50°C, 30 sec; and 72°C, 1 min.
Developmental Observation and Phenotypic Analysis
The whole processes of embryonic development of common carp, goldfish, the hybrid fish, and the nuclear transplants were examined under an Olympus SZX-12 microscope. The serial timings of somite development were recorded. The adults of common carp, goldfish, the hybrid fish, and the cloned fish were sampled for phenotypic analysis. These fishes were immobilized in 80ppm MS-222 (Sigma) and x-ray photographed using Super Soft X-ray Inspection System (Model CMB-2; Softex Co., Tokyo, Japan). X-ray films were developed as described in the user's manual and scanned using a Microtek ScanMaker 4800i (Shanghai, China). The vertebral numbers were counted from the scanned pictures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Larval red common carp did not show any pigmentation (Fig. 1A), while larval goldfish did (Fig. 1B). Thus, pigmentation could serve as a marker to determine whether the red common carp nuclei contribute to the development of NT larval fish. In total, five batches of successful nuclear transplantations were conducted, in which 52.9% of 501 transplanted eggs developed to the blastula stage. However, 62.6% of these blastulae failed to develop further to gastrulation, which seems to be a critical stage in the development of NT embryos, just as the midblastula transition is crucial to normal development of fish [21]. In some cases, the NT embryos excluded a small proportion of recipient yolk during gastrulation (Fig. 1, C and D), but this occurrence did not interrupt the subsequent development. A total of 99 blastulae (19.8% of transplanted eggs) developed to the gastrula stage, of which 12 (2.4% of transplanted eggs) were hatched. All the hatched nuclear transplants did not show any pigmentation (Fig. 1E), indicating that the red common carp nuclei contributed to the development of NT fry. Among the hatched fry, three failed to reach the blood-circulation stage (Fig. 1F) and two failed to feed, while the remaining seven (1.4% of transplanted eggs) reached adulthood. When these cloned fish were put into the same tank with red common carp and goldfish, it was difficult to find any difference between the cloned fish and the red common carp that provided nuclei. The exterior phenotypic characteristics of red common carp, such as two pairs of barbells, long body shape, normal tail, and normal eyes were present in the cloned fish, but there was almost no visible contribution of distinctive goldfish characteristics, such as spherical body shape, triangle tail, and dragon eyes (Fig. 1G). None of the 2-yr-old cloned fish could produce sperm or mature eggs.
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Nuclear DNA Genotypes of Nuclear Transplants
PCR amplification showed that all of 50 randomly sampled NT embryos had the characteristic hGH-transgene band (data now shown). The transgene was also detected in all of the cloned fish, in line with transgenic common carp that provided the donor nuclei (Fig. 2A). In addition, a comparative RAPD assay was developed to distinguish common carp, goldfish, and hybrid fish. Among 20 oligonucleotide primers, four of them (S121, S123, S128, S136) could produce different and distinguishable patterns for common carp and goldfish to identify the origins of the nuclear genomes of the cloned fish. However, the RAPD pattern of the hybrid fish did not often present both bands of common carp and goldfish, which may be due to the recombination between the genomes of common carp and goldfish in the hybrid genome. We found that the RAPD pattern resulting from each primer of the cloned fish DNA was identical to those from common carp and distinctly different from those of goldfish and the hybrid fish (Fig. 2B). Both results of transgene-based PCR amplification and comparative RAPD analysis proved that nuclear DNA (nDNA) of the cloned fish was exclusively derived from common carp, the source of the transplanted nuclei.
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The mtDNA Genotypes of Nuclear Transplants
The mtDNA genotypes of the cloned embryos at different stages and the cloned fish were analyzed by PCR with two sets of species-specific primers. The result showed that each NT blastula contained a mixture of two types of mtDNA genome, one from goldfish and another from common carp. The amplified yields with carp-specific primers were tiny and varied among the embryos, while the amplified yields with goldfish-specific primers were abundant and uniform among the embryos (Fig. 2C). These data indicate that the NT blastulae were all mtDNA heterogeneous, containing abundant recipient-type mtDNA while harboring a relatively small amount of donor-type mtDNA. This may be due to variation in the mtDNA copy numbers that accompanied transplanted nuclei. In contrast, in late-stage NT embryos after blood circulation, only goldfish-derived mtDNA genotype could be detected (Fig. 2D). This suggests that the contaminating mtDNA from the nuclear donor cells was eliminated during development in the nuclear transplants. Similar results were found during the development of the hybrid fish. Common carp-derived mtDNA could be detected before the blastula stage but not in gastrula and the following developmental stages of the hybrid fish (data not shown).
Somite Development and Vertebral Number of Nuclear Transplants
From the view of somite development, the embryonic development rate of nuclear transplants was a little slower than that of the nuclear-donor species, common carp, but was similar to that of the recipient species, goldfish (Table 1). The somite number in the only NT embryo that developed to larval stage was 29, which is within the range of goldfish. Similar results were found among the hybrid embryos, which had somite numbers that ranged from 28 to 30. X-ray photographs showed that the vertebral number of six cloned fish was of the enucleated egg providing goldfish type, ranging from 26 to 28. Of all the seven cloned fish, the vertebral number was 26 for one fish, 27 for two fish, 28 for three fish, and 31 for one fish. For one of the survival hybrid fish, the vertebral number was 28. In contrast, the vertebral number of nuclear-donor common carp was 33– 36 (Fig. 3). These data suggest that the goldfish egg cytoplasm plays an important role in regulating the somite development and vertebral number in the nuclear transplants.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In mammalian cloning, studies on the mtDNA genotypes of cloned animals are quite controversial. Although the first somatic cloned mammal [26] and the cross-species somatic cloned mammals [8, 9] showed to be mtDNA homoplasmy that contained just recipient cytoplasm-derived mtDNA, some cloned mammals were found to be mtDNA heteroplasmy—they contained mtDNA representative of both the donor cells as well as the recipient eggs [27, 28] The present study demonstrates that the goldfish-derived mtDNA can exist in cloned embryos until the blood-circulation stage, after which it faded away. In other words, the mtDNA heteroplasmy in cloned embryos converted to mtDNA homoplasmy over the course of development. From this point of view, the cross-genus cloned fish could be properly referred to as a nucleo-cytoplasmic hybrid fish that contains a combination of common carp-derived nuclear genome and goldfish-derived mitochondrial genome. However, the mechanism underlying the absence of donor cell-derived mtDNA in most cloned animals was unclear. In the present study, the mtDNAs from the nuclear donor cells were eliminated during the development of the cloned fish, just mimicking the destiny of the sperm-derived mtDNA in the sexual hybrid fish. In addition, the cross-genus cloned fish is as healthy as their peers of nuclear donors. This indicated that goldfish-derived mtDNA could not only work together with common carp nDNA but also be responsible for all the deserved pathways, though mitochondria control many fundamental metabolic pathways [29], just as nonhuman primate-derived mtDNA was able to survive in mtDNA-less human cells [30].
In previous studies of animal cloning, as expected, most cloned animals were identical to their nuclear donor species in phenotype [3–8]. Likewise, in the present study, most development characteristics of the cloned fish were the same as those of nuclear donor common carp. But, strikingly, analysis of somite development and vertebral number led to an unexpected result: vertebral development resembled that of the cytoplasmic recipient. In ongoing studies of reciprocal NT, the cloned fish need to grow bigger for analysis of vertebral numbers. Until now, the mechanism of vertebrate somitogenesis has been a mystery, although several theoretical models have been proposed [31]. The segmentation clock is the essence of many recently proposed models, which could explain most aspects of somitogenesis, such as the variation of somite numbers [32, 33]. However, little is known about the clock, especially the mechanism that drives it [33]. According to our data, the clock is likely regulated by cytoplasmic factors in the egg cytoplasm. This resulted in vertebral numbers in most cloned fish that were the same as those of egg-donating goldfish and different from that of nuclear-donor common carp, based on the concept that the vertebrae can be aligned with somite in vertebrates [34]. In previous studies, most embryonic induction factors are expressed in the maternal body and accumulated in mature oocytes [35]. Our data suggest that the somite number or segmentation clock of fish is determined in early embryogenesis under the regulation of egg cytoplasmic components during the formation of presomitic mesoderm [36]. During the somite stages, somitogenesis-related genes are expressed cyclically, resulting in the formation of somites [33]. Meanwhile, as a result of hierarchical activation of cascades of nuclear genes, cell fates and most of the developmental characteristics of fish are controlled by the temporal and spatial expression of nuclear genes. Therefore, the cross-genus cloned fish showed to be almost the same as donor common carp in exterior appearance.
In addition, in the studies of fish gene transfer, foreign gene integration occurs gradually and randomly in host genomes, which results in many problems, such as multi-site integration, positional effects, and transgenic mosaicism [11, 19, 37]. Here, the first case of successful cloning with nuclei from valuable, fast-growing GH-transgenic fish has been demonstrated. This is a crucial step for further cloning with in vitro genetically modified, cultured cells—a step that is considered to be an efficient method to solve these problems [11, 38]. On the other hand, some species of fish are near the edge of extinction due to environmental pollution and overfishing [39]. The present study provides a promising way to preserve endangered fish species through cross-species cloning by transplanting the nuclei of the endangered species into the enucleated eggs of another well-populated species.
Overall, the present study reveals that goldfish enucleated eggs could not only support the development of the cross-genus nuclear transplants receiving common carp nuclei but also have an evident impact on certain developmental characteristics, especially the somite development and vertebral number of the nuclear transplants.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 86 27 6878 0628; zyzhu{at}ihb.ac.cn
Received: 29 April 2004.
First decision: 24 May 2004.
Accepted: 21 September 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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