Laboratory of Theriogenology, Department of Veterinary Science, Yamaguchi University, Japan
The present results suggested the feasibility of enhanced green fluorescent protein (EGFP) gene for non-invasive selection of transgenic bovine embryos at the pre-implantation stage by using the fluorescent microscope and linking the marker gene contacted with a desired gene. However, these injected embryos showed impaired development and high rate of the mosaic expression. Therefore, we conducted a trial to transfer the EGFP gene fragment into the bovine or cat foetus fibroblasts using polybrene for expressing bright green fluorescence in the whole inner cell mass. EGFP has a great advantage as a marker because the transgenic cells or organ can be observed at any time in their viable and intact state and it can be easily connected with pharmaceutical protein in vitro. Additional advantages of nuclear transfer combined with transgenesis become obvious when it is compared with microinjection techniques. In the future, we can produce the pharmaceutical proteins from cow or cat using nuclear transfer combining with the EGFP gene.
Keywords: Clone, embryo, gene expression, microinjection, transgenic animal
Transgenic livestock have been developed for a variety of purposes, including improvement of food products or disease resistance, production of valuable therapeutic products in milk, and as models of human diseases (Hennighausen 1990; Janne et al. 1992; Colman 1996; Echelard 1996; Young et al. 1997; Ziomek 1998). However, the cost of transferring microinjected embryos to recipients that do not generate transgenic offspring is a major constraint to this approach. Therefore, for the production of large transgenic animals such as cattle, detection of the transgene at the pre-implantation stage would be desirable if considering the long gestation period and limited number of offspring. In bovine studies, although several non-invasive methods using firefly luciferase (Menck et al. 1997; Murakami et al. 1998a; Nakamura et al. 1998) or neomycin resistance gene (Bondioli and Wall 1996) as markers have been reported for selection of the transgenic embryos, the former method requires a step for loading the substrate (luciferin) inside the cells which is known to be toxic, whereas the latter requires the presence of neomycin in the culture medium.
In the last few years, green fluorescent protein (GFP), a protein of 238 amino acids found in jellyfish, Aequorea victoria, has been applied for various objectives as a useful marker for monitoring gene expression in situ (Chalfie et al. 1994). GFP absorbs blue light and emits green fluorescence without any need for exogenous substrates or cofactors, and this characteristic is of great advantage for GFP as a marker. Since no preliminary steps are required for the detection of GFP, cells or organs can be observed at any time in their viable and intact form by simple use of a fluorescent microscope. Therefore, the present study was conducted as a preliminary experiment aimed at evaluating the applicability of this convenient marker for selection in vitro of transgenic bovine/cat embryos. The fluorescence by pre-implantation expression in the bovine/cat embryos was observed after pronuclear microinjection or cloning with an enhanced GFP (EGFP; S65T; +F64L) gene construct (Okabe et al. 1997).
The methods used for in vitro maturation, in vitro fertilisation (IVF), and subsequent cultures in the experiment were modifications described by Boediono et al. (1994). Briefly, cumulus-oocyte complexes (COCs) were aspirated from the follicles (2–7 mm in diameter) of cow ovaries collected at a local abattoir and cultured in maturation medium (25 mM Hepes TCM-199) with Eagle's salts (Gibco, Grand Island, NY) supplemented with 5% superovulated cow serum (SCS), 0.01 mg/mL follicle stimulating hormone (FSH, Denka, Kawasaki, Japan), 20 mM taurine (Wako, Osaka, Japan), and 50 mg/mL gentamicin (Sigma, St. Louis, MO) at 38.5°C under 5% CO2 in air. After 20–22 hours of culture, the COCs were fertilised in vitro with frozen-thawed sperm for 5 hours in Brackett and Oliphant medium (Brackett and Oliphant 1975) containing 2.5 mM caffeine (Sigma), 3 mg/mL bovine serum albumin (BSA, fraction V; Sigma), 20 mg/mL heparin (Shimizu Pharmaceuticals, Japan) and 20 mM taurine, and then cultured in vitro in TCM-199 medium supplemented with 5% SCS, 5 mg/mL insulin (Sigma), 20 mM taurine, and 50 μg/mL gentamicin (Sigma, St. Louis, MO) at 38.5°C under 5% CO2 in air.
After 17 hours of IVF, these presumptive zygotes were incubated with 300 IU/mL hyaluronidase (Sigma) for 20 minutes, and their adherent cumulus cells were removed by repeated pipetting. The denuded zygotes were centrifuged at 16,000 g for 20 minutes in the presence of 5 μg/mL cytochalasin B (Sigma) to visualise the pronuclei (Fahrudin et al. 2000), and then transferred to a 100 μL drop of Dulbecco's phosphate buffer saline without Ca2+ and Mg2+ [DPBS (–); Gibco] supplemented with 3 mg/mL BSA, 5 μg/mL cytochalasin B, and 50 μg/mL gentamicin. After transfer, the pronuclei of the zygotes were microinjected with approximately 2 μL of the buffer solution (10 mM Tris-HCL, 0.2 mM EDTA, pH 7.5) containing 1.5 μg/mL EGFP cDNA fragment under control of the chicken beta-actin promoter and cytomegalovirus enhancer (Okabe et al. 1997) using an injection capillary (0.5 mm in diameter) attached to the micromanipulator (Leitz, Wetzlar, Germany). These injected zygotes were cultured in vitro for an additional 8 days (until day 9; IVF = day 0) to examine their developmental competence and fluorescent expression.
The green-light emission by the injected embryos was detected under a fluorescent microscope with DM 505 filters (EX 450–490 and BA 520, Nikon, Tokyo, Japan). The developmental stage of the fluorescent embryos was recorded on every second day after injection until day 9.
The application of nuclear technology using blastomeres of early bovine embryos has been reported (Prather et al. 1987; Fahrudin et al. 2000). The donor nuclei were prepared from the 8-cell stage embryos, which were observed for fluorescent expression.
The recipient cytoplasts were prepared from in vitro matured oocytes, which were enucleated at 20–22 hours after the beginning of in vitro maturation. Pushing out the first polar body and a small amount of cytoplasm after cutting the zona pellucida with a sharp glass needle did the enucleation process. The successful enucleation was confirmed by culturing the oocytes in the medium containing Hoechst 33342 (2 μ/mL) for 20 minutes, and subsequently exposing to ultraviolet light for a few seconds. All manipulations were done in 20 μL drops of phosphate buffer saline (PBS) supplemented with 5 μg/mL cytochalasin B and 0.3% BSA covered by mineral oil.
The embryos were reconstructed by fusing donor cells and presumptive metaphase cytoplasts, the transfer and fusion were accomplished immediately after the enucleation of the metaphase plate of the recipient oocytes.
The fusion was initiated by a single DC pulse of 1 kv/cm for 50 μsec (delivered by BTX 2001, San Diego, CA) in Zimmerman medium. The fused couple were then perthenogenetically activated by exposing into culture medium (CR 1aa supplemented with 3% BSA and 5% foetal calf serum (FCS)) containing calcium ionophore (10 μg/mL) for 5 minutes followed by culturing in the medium containing cycloheximide (10 μg/mL) for 5 hours. After the activation, five to ten reconstructed embryos were cultured in 100 μL drop of culture medium covered by mineral oil at 38.5°C in humidified chamber with 5% CO2 in air for 3 days. The developmental stage of the fluorescent embryos was recorded on every second day after fusion.
The methods used for nuclear technology with foetus cell lines (Takada et al. 1997; Wilmut et al. 1997; Cibelli et al. 1998; Stice et al. 1998) and subsequent EGFP gene transfers in this experiment were modifications of previous reports (Okabe et al. 1997; Murakami et al. 1999).
The donor nuclei (somatic cells) were prepared from bovine and cat foetuses cell lines, which were derived from frozen-thawed foetuses that had been frozen at –20° for about three months. Seven to twelve passages of the cell lines were used as donor nuclei in this study.
We conducted the transfer of the EGFP gene fragment into the bovine or cat foetus fibroblasts using polybrene or electroporation as follows:
The cells were in DME medium supplemented with 0.5% serum for 4–5 days before being used as donor nuclei. The cloning process and observation of fluorescent expression of embryos were done in the same way as experiments 1 and 2. The developmental stage of the fluorescent embryos was recorded on every second day after injection until day 9. However, the numbers of fluorescent embryos that were observed to include non-fluorescent blastomeres were recorded as the criteria of mosaic expression, regardless of the brightness of light-expression by the embryos.
The rate of development to blastocysts and expanded/hatched blastocysts was compared between the gene-transfer and non-treatment groups, and the rate of fluorescent expression was compared between the whole and mosaic groups using the Chi-Square (c2) test.
In the first experiment, a total of 310 zygotes were microinjected with the EGFP gene construct into the pronuclei. As the first step, the developmental competence in vitro of the gene-injected embryos was examined, and the results were summarised. Forty six (14.8%) zygotes degenerated within 24 hours after injection. The cleavage rate for the injection group was lower than that of the non-treatment group. However, statistical comparison between the two groups was not made because the oocytes in the non-treatment group had not been selected for the presence of pronuclei. The rate of development to blastocysts was calculated for the cleaved embryos in order to facilitate the comparison between the injection and non-treatment groups. The rate of development to blastocysts in the injection group was significantly lower (P<0.01) than that of the non-treatment group. Among these blastocysts, the rate of development to expanded/hatched blastocysts was lower (P<0.05) in the injection group than the non-treatment group, 56.8% (25/44) and 73.3% (121/165), respectively.
After gene-injection, the fluorescent expression was observed in a total of 37 (11.9%) embryos. However, although the fluorescent embryos at the blastocyst stage were detected in 2.9% of the injected embryos, six out of nine fluorescent embryos (66.7%) showed mosaic expression in their inner cell mass and trophectoderm. Furthermore, the rate of fluorescent embryos that showed mosaic expression after injection was significantly higher (P<0.05) than the embryos of whole expression (8.4% and 3.5%, respectively).
The developmental competence of bovine zygotes decreased after pronuclear microinjection with the EGFP gene. The centrifugation treatment for the polarisation of intracellular lipids, as performed herein, has been shown to maintain the normal developmental capacity in vitro of bovine zygotes in our previous experiment (Murakami et al. 1998b). It has been demonstrated that pronuclear microinjection with either water or buffer decreased bovine embryonic development significantly, and inclusion of DNA in the injection buffer decreased the development even more drastically (Peura et al. 1995). Therefore, the developmental restriction of zygotes that was found in this experiment was considered attributable to the treatment due to mechanical damage resulting from embryo manipulation; influence of the microinjected DNA itself could not be determined. In addition, the frequent observation of the embryos under the excitation light might have detrimental effect on their developments.
Previous studies have shown that the expression of EGFP was detected in preimplantation bovine embryos following pronuclear microinjection with the modified GFP (S65T) gene constructs (Chan et al. 1997; Takada et al. 1997). However, it has been indicated that some GFP-positive morale became weakly positive or almost negative at the blastocyst stage (Takada et al. 1997). In the present study, such a loss in fluorescent expression was not detected in the light-emitting embryos during the observation carried out after injection of the EGFP (S65T + F64L) gene. On the contrary, some non-fluorescent morale were observed to emit green-light when they developed to the blastocyst stage, 0.6% (2/31). We could not clarify the cause of the difference in fluorescent expression by the embryos. However, in the study of EGFP transgenic mouse (Okabe et al. 1997), humanised modification of the codon usage in the EGFP sequence was assumed to be responsible for the increased efficiency of their fluorescent expression.
The fluorescence was expressed by the injected embryos at various intensities throughout their development up to blastocyst stage. However, only 1.0% of the injected embryos developed to blastocysts that emitted green-light in their whole blastomeres. Moreover, the majority of the fluorescent embryos showed mosaic expression, 70.3% (26/37). It has been indicated that the mosaic was detectable commonly in approximately half of the fluorescent embryos when the EGFP gene construct used herein was injected into mouse zygotes (M. Okabe, personal communication). Although the exact reason for this phenomenon is unknown, Lewis-Williams et al. (1996) has demonstrated that the incidence of transgenic mosaicism in mouse embryos increased significantly with time after microinjection of a target DNA, using fluorescence in situ hybridisation (FISH) analysis. They indicated that most integration events seem to occur after the first cleavage, generating a majority of mosaics among the transgenic offspring. It has also been revealed that delayed integration of microinjected DNA into the embryo genome often results in mosaic founder animals (Schnicke et al. 1997).
In experiment 2, the rate of survival and fluorescent expression of the reconstructed embryos were 69.45 (25/36) and 52% (13/25), respectively. These results indicate that the rate of expression of EGFP gene with the nuclear transfer embryos is higher than that of microinjected embryos. In experiment 3, the results were almost the same as those of experiment 2. EGFP gene will be linked with any pharmaceutical protein during culture with fibroblast cells using polybrene or electroporation procedure. What genes should be added or removed? One gene of interest is the rhAT gene, which is a factor of releasing solidification of blood for human.
Additional advantages of nuclear transfer combined with transgenesis become obvious when it is compared with microinjection transgenesis. One advantage is the time and expense saved with nuclear transfer approach. With nuclear transfer all of the initial offspring are transgenic because the gene of interest is inserted into the fibroblast cells prior to making the nuclear transfer embryos. Then only nuclei from transgenic cell lines are used in the process. Using nuclei from lines of cells in which the sex is known, the sex of the transgenic offspring can be predetermined (in this case as female). This alone saves two years or several months of development time by eliminating one generation of cattle or cat, respectively. Since these are all genetically identical females, testing for expression and herd expansion can be performed rapidly.
In the present study, we used the cat for gene transfer by cloning technology. This technology is very efficient because the gestation period of cat is only 60 days. Therefore, the isolation of the efficiency protein from milk should lead to a safer and more cost-effective product.
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