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Department of Developmental Biology,4 Division of Insect and Animal, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-0901, Japan
Prime Tech Ltd.,5 Tsuchiura, Ibaraki, 300-841, Japan
The Institute of Medical Sciences,6 Tokai University, Bohseidai, Isehara, Kanagawa, 259-1143, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Puromycin is an antibiotic that inhibits growth of animal cells and blocks protein synthesis by binding to 80S ribosomes at low doses. The puromycin-resistant gene (termed pac) encoding puromycin N-acetyl transferase was isolated from Streptomyces aboniger [9]. If pac is introduced and expressed in animal cells, the cells can survive in the presence of puromycin [10]. Our previous report [11] demonstrated that puromycin killed wild-type embryonic stem (ES) cells after short periods of exposure (2 days) in mice. Furthermore, puromycin was effective at a very low concentration of 1 µg/ml. It generally takes only 7 days to obtain stable transfectants. On the other hand, G418 has been widely used for the selection of various cells after transfection [12]. In stark contrast with puromycin, G418 requires 10–14 days to obtain transfectants and much higher concentrations (100–1500 µg/ml) [7] are required to kill nontransduced cells.
In this study, we investigated effects of puromycin for the selection of transduced cells to use in porcine nuclear transfer experiments. Somatic cells primarily isolated from porcine fetuses were directly subjected to electroporation with an enhanced green fluorescent protein (EGFP) expression vector containing pac. These electroporated cells then underwent selection with puromycin for 7 days. After selection, EGFP-expressing transfectants were directly subjected to nuclear transfer in pigs. As a result, we obtained one healthy piglet expressing EGFP systemically. The significance of our simplified method for generating transgenic pigs via nuclear transfer is discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Construction of Vectors
The transgene, termed pEGFPac (Fig. 1), was constructed by inserting CAG-EGFP fragment into pPGK-pac-p(A) cassettes. Briefly, pCAG-EGFP, an EGFP-expression cassette, was first constructed according to Okabe et al. [13]. The EGFP cDNA was PCR amplified from pEGFP-C1 (Clonetech, Palo Alto, CA) using F1 (5'-ttgaattcgccaccatggtgagc-3') and R1 (5'-ttgaattcttacttgtacagctcgtcc-3') primers. After digestion of the amplified cDNA fragments with EcoRI, these fragments were subcloned into the EcoRI site of the pCAGGS expression vector [14] that contains the cytomegalovirus enhancer, chicken ßactin promoter, and a portion of second intron, third exon, and 3' noncoding region of the rabbit ßglobin gene. The PGK-pac-p(A) cassette obtained from digesting pPGK-pac-p(A) [11] with SalI, was introduced into the PstI site of pCAG-EGFP by blunt-end ligation. The resulting plasmid was termed pEGFPac, and sequenced to check that the insert had been properly inserted. A 4.8-kilobase (kb) fragment containing EGFPac was isolated, before gene transfer, from the SalI-digested pEGFPac after electrophoresis on a 0.8% agarose gel.
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Isolation of Fetal Somatic Cells
A pregnant female sow (Landrace) was killed at 73 days, and fetuses were dissected out. The associated yolk sac and amnion were removed from the fetuses under germ-free conditions. After washing with Dulbecco modified phosphate buffered saline without Ca2+ and Mg2+ [PBS(–)], two fetuses were minced using scissors and dispersed by incubating them at 37°C for 20 min in 10 ml of PBS(–) containing 1% (w/v) of collagenase (C9407; Sigma Co. Ltd., St. Louis, MO) and 0.1% (w/v) of trypsin (159090-46; Gibco, Grand Island, NY). Dissociated somatic 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. A small portion of cells was subjected to a puromycin sensitivity assay as described below. The remaining cells were immediately subjected to electroporation-mediated gene transfer as described below, and the remaining cells were deep frozen using culture medium containing 10% (v/v) dimethyl sulfoxide (DMSO) (D2650; Sigma Co. Ltd.).
Puromycin Sensitivity Assay
Stock solution (10 mg/ml) of puromycin (P8833; Sigma Co. Ltd.) was prepared by dissolving puromycin in distilled water at the appropriate concentration and stored at 4°C. Media containing variable amounts of puromycin were freshly prepared by adding the appropriate volume of puromycin stock solution.
To determine the optimal concentration of puromycin for selecting EGFPac-transfected cells, a puromycin resistance test was performed with fetal porcine somatic cells. Cells were seeded in 24-well plates at a density of 2.5 x 104 cells per well and cultured in medium containing 0.5–6 µg/ ml puromycin for 7 days. A total of 12 wells containing puromycin at indicated concentrations were used per assay. This assay was repeated three times. Cell viability was examined by using the Cell Titer-Glo Luminescent Cell Viability Assay Kit (G7571; Promega Co., Madison, WI). Cells from 12 independent wells were separately measured for luciferase activity. Luciferase activity was measured using a luminometer (CT-9000D; Dia-Iatron, Tokyo, Japan) following the protocols described by the manufacturer. Because there is a possibility that each well might contain a variable number of cells, data on luciferase activity were normalized against the protein concentration in each well. The resulting data on 12 independent cells were statistically analyzed using a two-tailed Student t-test. Based on these treatments, cell viability was plotted and analyzed.
The survivability of primary-cultured fetal porcine somatic cells was also examined by culturing in a medium containing 2 µg/ml of puromycin for up to 7 days. Cells were harvested at 2–7 days after treatment with puromycin and inspected for cell viability as described above. Each experiment was performed three times. The number of viable cells was expressed as percentage ± standard error.
Gene Transfer and Selection of Transfectants
Fleshly isolated fetal somatic cells (1 x 107) were electroporated in 500 µl of HEPES-buffered saline containing 10 µg of EGFPac using an electroporation system (Gene Pulser II; Bio-Rad Co. Ltd., Hercules, CA) with 750 V/cm, 950 µF one pulse and 40 msec wavelength. Electroporated cells were then cultured in a 100-mm plastic dish (#3003; Becton Dickinson, Franklin Lakes, NJ) with 10 ml of Dulbecco modified Eagle medium (DMEM) (D5796; Sigma Co. Ltd.) containing 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 in air. After 48 h in culture, 2 µg/ml of puromycin was added to the medium for selection of cells carrying EGFPac. On the seventh day following selection, surviving cells were grown to confluence and the expression of EGFP was confirmed under a fluorescence microscope as described below. These cells were then transferred into a 60-mm dish (#3004; Becton Dickinson) for nuclear transfer, and the remaining cells were deep frozen using culture medium containing 10% (v/v) DMSO. In total, it took 7 days to obtain the recombinant donor cells. As a control group, freshly isolated fetal somatic cells (1 x 107) were cultured for 2–3 days until confluent and then electroporated in 500 µl of HEPES-buffered saline containing 10 µg of EGFPac as described above. The colonies of the recombinant fibroblast cells were transferred into 24-well plates after EGFPac transfections and puromycin selection. These cloned cells were then cultured for 3–4 days until confluent. Finally, these cells were transferred into a 35-mm dish and subcultured for 3–4 days until nuclear transfer. In this group, the total culture period of the recombinant cells was around 16–18 days. Also, freshly isolated fetal somatic cells that had not been subjected to electroporation were also cultured for 2–3 days and then subjected to nuclear transfer or observation for EGFP fluorescence as a positive control for nuclear transfer and as a negative control for EGFPac transfections, respectively.
Nuclear Transfer and Transplantation of Manipulated Embryos to Recipients
Transgenic cloned piglets were produced by nuclear transfer as described previously [15]. Briefly, nuclei from puromycin-selected transfectants or normal fetal somatic cells were each introduced into a single enucleated oocyte by piezo-actuated microinjection. A total of 3212 enucleated oocytes were subjected to nuclear transfer with nuclei from the puromycin-selected transformants. Similarly, a total of 250 enucleated oocytes were transplanted with nuclei from wild-type cells. The reconstructed (nuclear-transplanted) oocytes were then electrically activated and cultivated in PZM3 medium [16] in an atmosphere of 5%CO2, 5%O2, and 90% air at 38.5°C for 2 days until two- to eight-cell stage. The surviving embryos were transferred to the oviducts (30–120 embryos per a surrogate) of an anesthetized surrogate mother (matured LWD; Landrace x Large White x Duroc crosses). A portion of the two- to eight-cell stage embryos were cultivated for 6 days until blastocyst stage and then inspected for EGFP fluorescence, as described below. Embryo transfer was performed nine times using 14 surrogates for the embryos derived from nuclear transplantation of puromycin-selected transfectants. Eleven surrogate mothers were used for embryo transfer of the embryos derived from nuclear transplantation of wild-type cells. These surrogate mothers were previously pseudopregnant by artificial insemination after injection of superovulation-inducing hormones and artificially aborted. After embryo transfer, these mothers were kept under a conventional environment employed for housing pigs and observed daily for confirmation of pregnancy by checking estrus. All of the cloned piglets were delivered by natural birth without any chemical induction.
Observation of EGFP Expression
Expression of EGFP in surviving cells after puromycin selection and nuclear transfer-derived blastocysts was examined under a fluorescence microscope (Olympus, Tokyo, Japan) with DM filters (Olympus). Expression of EGFP in the surface skin of cloned piglets was examined visually using Orange Spectacles (UVP, Upland, CA) under an appropriate excitation light source (wavelength 480 nm) converted from ultraviolet (UV) light (302 nm) using a UV/Blue converter plate (UVP) or in cells isolated from tissues such as the ear under a fluorescence microscope as reported by Okabe et al. [13]. Photographs were taken under the excitation light source with a Sharp Cut Filter (Y-50; Kenko, Tokyo, Japan) using a NIKON D1H digital camera (Tokyo, Japan). No image intensifying procedure was applied to any of the photos.
Transgene Analysis
Genomic DNA was extracted from the primary-cultured somatic cells after gene transfer; tissues (including ear and liver) were obtained either by biopsy from live piglets or from animals that had died shortly after birth, as described by Blin and Stafford [17].
For PCR analysis, 1 µl of DNA (0.1 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 sec, 58°C for 25 sec, and 72°C for 3 min (30 cycles). Primers used were EGFPf2 (5'-gacgtaaacggccacaagttc-3') and EGFPr2 (5'-atgccgttcttctgcttgtc-3') (Fig. 1). This primer set yields a 421-base pair (bp) product; products were analyzed by electrophoresis on a 1.5% agarose gel and visualized under UV illumination after staining gels with ethidium bromide. One nanogram of pEGFPac vector DNA was used in the PCR reaction as a positive control. For the negative control, 1 µl of genomic DNA (0.1 µg) isolated from Landrace liver was also subjected to PCR. 
x 174 phage DNA digested with HincII was used as molecular weight markers.
For genomic Southern blot analysis, genomic DNA (10 µg) was digested with EcoRI, thereby producing a 0.7-kb fragment containing the EGFP cDNA sequence. After electrophoresis of the enzyme-digested DNA on a 1.0% agarose gel, the DNA was finally transferred onto Hybond-N+ filters (Amersham Pharmacia Biotech, Little Chalfont, England). These filters were hybridized with a 32P-labeled EGFP cDNA probe, then exposed to an Imaging Plate (Fuji Film, Tokyo, Japan) overnight, and finally analyzed with the aid of a FLA-3000G image analyzer (Fuji Film).
Western Blot Analysis
Puromycin-selected transfectants and primary cultured cells derived from cloned transgenic piglets were homogenized in sample buffer (10% sucrose [w/v], 3% SDS [w/v], 60 mM Tris-HCl [pH 6.8)]). After the supernatants were treated with ßmercaptoethanol, they were separated by electrophoresis under reducing conditions on a 10% polyacrylamide-SDS-gel and transferred to nylon membranes (Immobilon-P; Millipore, Bedford, MA). Blots were 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 anti-GFP antibody (ABR, Golden, CO). After washing with TBS, blots were incubated with horse radish peroxidase (HRP)-linked anti-rabbit IgG in TBS containing nonfat dry milk. Blots were then rewashed with TBS. EGFP proteins were detected by treatment of the membranes with ECL Plus western blotting reagent (Amersham) and subsequent exposure to an x-ray film for several minutes at room temperature.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the optimal concentration of puromycin for the selection of pac-transfected cells, a puromycin resistance test was performed by culturing fetal porcine somatic cells in a medium containing 0.5–6.0 µg/ml of puromycin for 7 days. No surviving colonies were obtained when exposed to puromycin at concentrations higher than 2 µg/ml (Fig. 2A). When cells were grown in medium containing 2 µg/ml of puromycin, none of the resultant cells survived for more than 6 days (Fig. 2B). We therefore used 2 µg/ml of puromycin for the selection of cells transfected with EGFPac.
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Isolation of Puromycin-Resistant Pig Somatic Cells
Approximately 104 cells survived in the presence of puromycin (2 µg/ml) after gene transfer involving the EGFPac construct. Almost all of these surviving cells exhibited EGFP fluorescence (Fig. 3, A and B). A portion of cells (a total of eight colonies) that had survived after selection with puromycin was then examined for the presence of EGFP cDNA by PCR; the EGFP cDNA was detected in each sample (data not shown). Western blotting analysis using an anti-GFP antibody also confirmed that these surviving cells expressed EGFP protein (which was identified as a 38-kDa band) (data not shown).
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Production of Transgenic Somatically Cloned Pigs
To obtain somatic clones in pigs, we transferred the nuclei of EGFP-expressing cells that had been selected with puromycin after gene transfer of EGFPac, to the enucleated oocyte. From a total of 3466 enucleated oocytes receiving nuclei from EGFP-expressing cells, 3212 oocytes survived, and 57.5% (1737) of these surviving oocytes developed in vitro to the normal two- to eight-cell stage (Table 1). When a portion of these developing two- to eight-cell stage embryos were cultured up to the blastocyst stage and then inspected for EGFP fluorescence, all embryos tested (5/5) exhibited bright fluorescence over the entire surface of the blastocyst (Fig. 3, C and D). When a total of 1401 enucleated oocytes were implanted with nuclei from untransfected fetal fibroblast cells, 1273 oocytes survived, and 43.0% (548) of these treated oocytes developed in vitro to the normal two- to eight-cell stages (Table 1). However, none (5/5 tested) of the blastocysts developed in vitro from the surviving two- to eight-cell embryos exhibited EGFP fluorescence (data not shown).
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From a total of 1680 developed embryos that had been derived from the nuclear transfer of puromycin-resistance cells transferred to 14 surrogate mothers, nine piglets (0.52%) were successfully delivered (Table 1). All of these piglets clearly expressed EGFP throughout their body surface when inspected under UV illumination (data not shown). Unfortunately, eight of the nine piglets died shortly after birth due to unknown reasons, but the remaining male piglet (termed L15-112) survived and appeared healthy. No histological and anatomical abnormalities were noted in any of the dead piglets (data not shown). The L15-112 piglet is now 10 mo old and still appears normal. In the control experiment, four (1.87%) piglets were obtained after transfer of a total of 213 developed embryos, which had previously been subject to nuclear transfer with nuclei of untransfected fetal fibroblast cells, to 11 surrogate mothers (Table 1). Unfortunately, one of the four piglets died accidentally, but the remaining piglets (termed M12-86, M13-11, and M14-42) grew healthily. On the other hand, no piglet was obtained from EGFPac recombinant cells by using the conventional cellular cloning before and after gene transfection (Table 1).
The surviving L15-112 pig (aged 10 mo) clearly expressed the EGFP in a variety of tissues. For example, intense fluorescence was observed in the skin (including hair) (Fig. 4, A and D), and epithelial tissues taken from the snout, oral, nasal mucosa, coronary band, and hoof wall (Fig. 4, E and F) under an excitation light source. To confirm the presence of the EGFPac transgene in somatically cloned pigs, genomic DNA prepared from the ear of live (L15-112) or dead (pig 1, pig 2, and L15-113: from the same litter as L15-112) piglets were subjected to PCR. All the samples tested contained EGFPac (Fig. 5). Southern blotting hybridization analysis also revealed the presence of a hybridization signal in all of the samples tested except from pig 5 (Fig. 6). Genomic DNA was degenerated in pig 5 and could not be obtained from pig 9 because of degradation. The transgene copy number was estimated to be 80 in the L15-112 piglet.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A long-term culture of primary cultured cells has been thought to cause cellular aging that leads to a reduction in the proliferative activity of cells [18]. Nuclei of aged cells have been associated with reductions in developmental potentiality in somatic cell cloning systems [8]. In this context, our present strategy, employing direct gene delivery to freshly isolated cells and short-term selection of transfected cells in the presence of puromycin, would clearly overcome these earlier problems. To our knowledge, there are very few reports concerning the successful gene delivery to cells freshly isolated from fetal tissue. This may be due to the widely accepted concept for a transfection protocol that exogenous DNA is easily introduced to rapidly dividing cells [19]. However, we observed a highly efficient gene transfer rate (1/50) into isolated fetal cells using an electroporation method. The method of electroporation-mediated gene transfer to freshly isolated (but not cultured) cells reduces time and cost and appears to be a powerful tool in various fields related to gene delivery to cells. Furthermore, employment of puromycin as a means of drug selection appears to be of importance for the systematic production of transgenic somatically cloned animals. This is solely because puromycin is effective at very low concentrations (>2 µg/ml) (see Fig. 2), even in slowly dividing cells such as cells from newborn piglets. If puromycin is added to more actively dividing cells, which synthesize proteins actively, it appears to block the growth of cells at concentrations lower than 2 µg/ml. The culture period required for obtaining stable transfectants after gene transfer and selection with puromycin is only 7 days. This appears also to be important in shortening the culture period because selection of G418, which is most widely used as a selection drug, requires at least 10 days to obtain stable transfectants [1–5]. Because the selection period of somatic cells influences success rate in the somatic cloning system [8] [20], we believe that puromycin is one of the best selection drugs to be reported thus far. Puromycin and G418 each work in an independent manner in mice ES cells [11]. If this is also true in pigs, then it follows that puromycin and G418 may be used as selection drugs to obtain double-knockout somatic cells in which a certain target locus is disrupted by insertion of the neocontaining gene construct and the corresponding allele by the pac-containing gene construct. Double-knockout cells would allow us to produce null-mutant pigs at founder level using the somatic cell cloning technique. Recently, Sharma et al. [21] reported a method for the successful production of double-knockout somatic cells. Briefly, they disrupted both alleles of the alpha 1,3-galactosyltransferase gene (1,3-GTase) in cultured somatic cells using a conventional gene targeting technique and produced somatic cell-derived cloned fetuses using the targeted cells. After the resulting fetuses were dissected out, primary-cultured fibroblastic cells were propagated in vitro and treated with anti-GTase monoclonal antibody [22] to eliminate cells expressing GTase and concentrate double-knockout recombinant cells. However, the efficiency of this method (which is called mitotic recombination) to obtain double-knockout cells was very low [23]. We believe that simultaneous use of both G418 and puromycin may greatly enhance the production rate of double-knockout somatic cells. Interestingly, Taniguchi et al. [24] demonstrated that puromycin is useful as a dominant marker for Cre recombinase expression in ES cells in the conditional gene targeting system. This indicates the potential usefulness of puromycin within the conditional gene targeting (and probably other fields related to gene engineering), as well as in somatic cloning system.
In the present study, we observed that most of our newborn piglets died postnatally. The reason for this high mortality is still unclear because histological and anatomical abnormality could not be found in these dead piglets. At present, there is little knowledge about the effect of puromycin N-acethyltransferase to early development of somatic cell clones in pigs. However, we believe that pac gene expression or puromycin selection itself is not deleterious to cell function because the EGFPac transfectants exhibited normal cell morphology, which was indistinguishable from that of untransfected cells, and exhibited EGFP without change in fluorescent strength. There is, however, a possibility that EGFP protein expressed in fetal piglets after nuclear transfer may be deleterious to postnatal development. Arat et al. [25] reported detrimental effects of the GFP protein during the somatic cloning procedure in cattle. These authors report that donor cells expressing GFP protein had reduced developmental ability when compared with nontransfected ones after nuclear transfer. These results indicated the harm that GFP protein may cause during early embryo development. Alternatively, abnormal genomic imprinting or epigenetic alteration in host chromosomal DNA, which probably generates during the nuclear transfer procedure, may result in the postnatal death of cloned piglets. Further research will be needed to make this point clear.
We possess a living transgenic cloned piglet (L15-112), aged 10 mo; he has no behavioral or metabolic abnormality at the present time. We are planning to obtain F1 offspring from L15-112, and using these offspring, we plan to address those issues mentioned above in more detail.
In conclusion, puromycin is useful as an effective drug for the selection of recombinant cells that will be used as donor cells in the production of somatically cloned gene engineered animals. Furthermore, we demonstrated that the direct gene transfer to freshly isolated porcine fetal cells is possible using electroporation. This method does not require short-term cultivation of cells which is always needed for the standard gene transfer protocol targeted to primary-cultured cells, and would accelerate production speed for transgenic somatic cell clone piglets by nuclear transfer. At present, however, this method is neither rapid nor convenient but does provide a novel means of producing transgenic farm animals using cloning technology. Moreover, the present study provides the foundation for the development of further methods.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Satoshi Watanabe, Department of Developmental Biology, Division of Insect and Animal, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukuba, Ibaraki, 305-0901, Japan. FAX: 81 29 838 8635; kettle{at}affrc.go.jp
3 Current address: Department of Research Planning and Coordination, National Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan
Received: 3 May 2004.
First decision: 24 May 2004.
Accepted: 3 September 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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