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Production of transgenic chickens from purified primordial germ cells infected with a lentiviral vector
Journal of Bioscience and Bioengineering VOL. 109 No. 4, 315 – 321, 2010 www.elsevier.com/locate/jbiosc
Production of transgenic chickens from purified primordial germ cells infected with a lentiviral vector Makoto Motono, Yuki Yamada, Yuki Hattori, Ryo Nakagawa, Ken-ichi Nishijima,⁎ and Shinji Iijima Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan Received 19 August 2009; accepted 6 October 2009 Available online 29 October 2009
Replication-defective retroviral or lentiviral vectors have been used for the production of transgenic animals. Chicken primordial germ cells (PGCs) are the precursors for ova and spermatozoa. Here, we describe the production of transgenic chickens via a germline transmission system using PGCs infected with a replication-defective lentiviral vector. PGCs were sorted with a fluorescence-activated cell sorter based on the expression of stage-specific embryonic antigen-1 from 2.5- and 5.5-day embryos. PGCs from both stages of embryo were infected with a lentiviral vector at a similar efficiency in vitro. PGCs were then transferred into the bloodstream of 2.5-day recipient embryos. The efficiency with which the PGCs were delivered and settled in the gonads was lower for PGCs from 5.5-day embryos than those from 2.5-day embryos when a limited number of PGCs was transferred, while the difference was not obvious upon the transfer of increased number of cells. Using a high number of 5.5-day PGCs infected with a lentiviral vector, transgenic chimeras (G0) with an acceptable efficiency for germline transmission were obtained. G0 female chickens produced transgenic progeny (G1) with higher efficiency compared to G0 male chickens. In G1 transgenic chickens obtained by this method, enhanced green fluorescent protein was effectively expressed under the control of the actin promoter. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Transgenic chicken; Transgenic avian bioreactor; Lentiviral vector; Primordial germ cell; Cell sorter]
Transgenic animals have been generated for the production of recombinant proteins in the milk of livestock species such as goats, sheep, pigs, and cows (1). However, mammalian transgenic systems have several drawbacks in that they require a relatively large area for breeding and a few years to become sexually mature. Therefore, avian species have attracted great attention as an alternative choice (2). In particular, chickens have several advantages such as high protein productivity in eggs, the small space required for breeding, and the lack of prion diseases. In addition, the N-glycosylated carbohydrate of chicken has N-acetylneuraminic acid at its end as in that of human (3). Several researchers have produced transgenic chickens by injecting viral vectors into embryos (4-14). Retroviral vectors derived from avian leukosis virus, reticuloendotheliosis virus, or Moloney murine leukemia virus have been injected into the subgerminal cavity at the blastodermal stage or into the heart of developing embryos to obtain transgenic chickens (4-11). Recently, lentiviral vectors have been used to produce transgenic chickens since they are relatively resistant to gene silencing (12-14). We and other research groups have reported on the production of recombinant proteins into eggs using genetically manipulated chickens (7-12), but in all cases, germline transmission efficiency is relatively low, which must be overcome for the practical application of transgenic technology in chickens.
⁎ Corresponding author. Tel.: +81 52 789 4279; fax: +81 52 789 3221. E-mail address: [email protected] (K. Nishijima).
Primordial germ cells (PGCs) are progenitor cells of ova and spermatozoa. Chicken PGCs can be recognized at the center of the area pellucida in the stage X blastoderm (freshly laid eggs). After gastrulation, they can be found in the extraembryonic region, the germinal crescent. They then enter and circulate in the blood vessels of the embryo, and migrate to the gonadal ridges, which develop into the mature gonads (15). Previous experiments showed that isolated PGCs could be transferred to other recipient embryos at the stages in which PGCs circulate in the blood, without losing the ability to contribute to the formation of germ cells (16-19). Thus, it is assumed that gene transfer to PGCs and subsequent transplantation to recipient embryos is an alternative method to generate transgenic chickens. Furthermore, it is expected that the use of PGC may improve the efficiency of germline transmission since PGCs are progenitors of ova and spermatozoa. In the early 1990s, it was shown that transgenic chickens could be established with PGCs infected with spleen necrosis virus, but a detailed analysis was not reported (20). Last year, transgenic quails were established using PGCs, but the efficiency was low (21). In addition, transgenic chickens could be obtained by transferring PGCs that had been cultured for more than one month, which permitted the selection of cells containing an integrated form of transgene (22). However, long-term cultivation of PGCs is difficult, and the efficiency of obtaining transgenic progeny varies across research groups possibly due to different experimental methods. One important factor determining the efficiency of obtaining transgenic progeny is the percentage of the transferred PGCs to the
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whole PGC population (transferred plus endogenous). Experiments in which PGCs were transferred without gene manipulation showed that the percentages of progeny from donor-derived PGCs varied depending on various experimental factors including the strain of chicken, source of PGCs, methods for PGC purification, and sex of birds (15-19). In these experiments, circulating PGCs (cPGCs) obtained from 2.5-day embryos were transferred to the blood vessels of the recipient embryos in the same developmental stage (17, 18). In addition, gonadal PGCs (gPGCs) from 5- to 5.5-day embryos were also used to produce germline chimeras despite the mismatch of their developmental stages (16, 19). However, direct comparison of the efficiency with which PGCs are delivered to the gonads of recipients has not been performed for cPGC and gPGC thus far. In order to optimize conditions for the establishment of transgenic chickens, we directly compared the efficiency of PGCs from 2.5- and 5.5-day embryos in migrating to the recipients' gonads and succeeded in establishing transgenic progeny using gPGCs infected with a lentiviral vector. MATERIALS AND METHODS Preparation of fertilized eggs Fertilized eggs were obtained by artificial insemination of White Leghorn (line M; originally obtained from Nisseiken, Yamanashi, Japan). The laid eggs were sterilized with 70% ethanol and were preserved at 12 °C until use. All experiments were performed according to the ethical guidelines of Nagoya University for animal experimentation. Preparation of cPGCs and gPGCs Fertilized eggs were incubated at 38 °C and 65% humidity with rocking every 15 min at an angle of 90°. To obtain blood cells, the sharp end of the eggs was cut horizontally after incubation for 51 h. After confirming that the embryos had developed to Hamburger–Hamilton stages 13–16 (23), blood was drawn with a glass micropipette from 30 to 40 embryos and resuspended in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, USA). The cells were centrifuged at 200 × g for 5 min at 4 °C and were washed with phosphate-buffered saline (PBS) containing 0.5% FBS. To obtain gonadal cells, embryos were isolated from fertilized eggs at day 5.5 of incubation (stages 27–28) and rinsed with PBS. Gonads were isolated with forceps and were dispersed in 0.1% trypsin and 0.02% EDTA. DMEM containing 10% FBS was added to stop trypsin digestion, and the cells were washed as described for blood cells. To purify cPGCs and gPGCs, dispersed blood and gonadal cells were incubated with 100 μl of a 1:100 dilution (in PBS containing 0.5% FBS) of anti-stage-specific embryonic antigen (SSEA)-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min on ice. After washing with PBS, 100 μl of a 1:200 dilution (in PBS containing 0.5% FBS) of phycoerythrin-labeled goat anti-mouse IgM antibody (Rockland, Gilbertsville, PA, USA) was added to the cell suspension and incubated for 30 min on ice. The suspension was washed with PBS and resuspended in 1 ml of PBS containing 0.5% FBS. Cells expressing SSEA-1 were sorted using a fluorescence-activated cell sorter (FACS) (EPICS ALTRA; Beckman-Coulter, Fullerton, CA, USA). The sorted SSEA-1-positive cells were resuspended in DMEM containing 10% FBS at a cell concentration of 100–200 cPGCs/ μl or 300–500 gPGCs/μl, based on cell counting by FACS. Plasmid construction Human immunodeficiency virus (HIV)-based lentiviral vector plasmid pSicoR (Addgene plasmid 11579) (24) was obtained from Addgene (Cambridge, MA, USA). It was digested with XbaI and NheI to remove the cytomegalovirus promoter, and the chicken β-actin promoter derived from pmiwZ (25) was inserted into the same restriction site to construct pLSi/ΔAeGFP, in which enhanced green fluorescent protein (eGFP) was expressed from the β-actin promoter (Fig. 1). Lentiviral vector production 293FT cells seeded on 24-well collagen-coated plates (Asahi Techno Glass, Chiba, Japan) were transfected with 300 ng of lentiviral
J. BIOSCI. BIOENG., vector (pLSi/ΔAeGFP), 100 ng of gag/pol plasmid (pLP1), 240 ng of rev plasmid (pLP2), and 160 ng of VSV-G plasmid (pLP VSV-G) (Invitrogen, Carlsbad, CA, USA) using Lipofectamine 2000 (Invitrogen). At 48 h posttransfection, the culture supernatant was collected and filtered through a 0.45-μm filter, followed by centrifugation at 20,000 × g for 5 h at 4 °C. The viral pellet was suspended in 7–10 μl of 50 mM Tris-HCl buffer (pH 7.8) containing 130 mM NaCl and 1 mM EDTA. Polybrene (Sigma-Aldrich) was added to the solution at a concentration of 8 μg/ml before the infection. The viral titer was determined with HeLa cells in terms of the percentage of cells expressing the eGFP gene. Viral infection in cultured PGCs The cPGCs and gPGCs were incubated with the lentiviral vector at a multiplicity of infection (MOI) of 30–100 in DMEM containing 10 % FBS for 2–4 h at 37 °C. The cells were then cultured on 96-well gelatin-coated microplates (Asahi Techno Glass) at 37 °C for 2 days in DMEM supplemented with 10% FBS, 10 mM HEPES (Nakarai Tesque, Kyoto, Japan), 1 × non-essential amino acids (Invitrogen), 10 ng/ml insulin-like growth factor-I (Invitrogen), 10 ng/ml basic fibroblast growth factor (Invitrogen), and 10 units/ml murine leukemia inhibitory factor (ESGRO; Chemicon International, Temecula, CA, USA) and were analyzed using FACS for the expression of eGFP. Analysis of transferred PGCs in the gonads of 9-day recipients To assess the percentage of donor-derived PGCs that had migrated and settled in the gonads of the recipients, PGCs derived from transgenic chickens producing single-chain antibody, established previously in our laboratory (7), were used as donor cells. Sorted PGCs were injected into the blood vessel of stages 13–16 recipient embryo. The sharp end of fertilized eggs that had been incubated for 51 h was cut horizontally to make an opening of 35 mm in diameter. To reduce the number of endogenous cPGCs in each recipient, the blood vessel of stages 13–16 recipient embryo was pricked with a fine glass micropipette, and 2–4 μl of blood was bled out as described previously (17). The sorted cells were then injected into the blood vessel of each manipulated embryo followed by insertion of bubbles to stop bleeding from the point of injection (26). The embryos were developed in surrogate chicken eggshells that had been sterilized by γradiation from 60Co prior to use. After incubation for 7 days, the recipient embryos were washed with PBS, and the gonads were dissected. The SSEA-1-positive cells were sorted by FACS and were preserved at -80 °C until analysis. In some experiments, the sex of recipient embryos was determined by morphological differences of the gonads, and each embryo was separately subjected to cell sorting. Genomic DNA was extracted from the sorted cells using a DNA preparation kit (QIAamp DNA Micro Kit; Qiagen, Hilden, Germany) according to the manufacturer's instructions. The percentage of donorderived cells that possessed the transgene was determined by real-time polymerase chain reaction (PCR) (LightCycler 1.5 Instrument; Roche Diagnostics, Mannheim, Germany) to amplify the retroviral vector sequence (packaging signal) using hybridization probes as described previously (7). The amount of genomic DNA was normalized by real-time PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH was amplified in 20 μl reaction mixture containing 10 μl Platinum SYBRGreen qPCR SuperMix-UDG (Invitrogen), 500 nM each primer, 1 μl 20 × bovine serum albumin (1 mg/ml), and 2 μl sample DNA with 45 cycles (94 °C for 5 s, 55 °C for 10 s, and 72 °C 10 s). The primers used were 5′-GGGCACGCCATCACTATC-3′ and 5′-GTGAAGACACCAGTGGACTCC-3′. Production of germline chimeric chickens The sorted gPGCs were incubated with the lentiviral vector at various MOI in DMEM containing 10% FBS for 2–4 h at 37 °C. To produce transgenic chimeras, the cells were injected into the blood vessels of stages 13–16 recipient embryos. One day before the expected day of hatching, rocking was stopped, and the plastic film was punctured to assist embryonic pulmonary respiration. Transgene detection by PCR Genomic DNA from the sperm was extracted using MagExtractor genome (Toyobo, Osaka, Japan). PCR was performed with 20 ng of the genomic DNA as a template. After initial denaturation of DNA at 94 °C for 1 min, PCR was started at 94 °C for 15 s, 58 °C for 30 s, and 68 °C for 15 s, for 35 cycles. The primers used were 5′-CGGCAACTACAAGACCCGC-3′ and 5′-GAAGTTCACCTTGATGCCGTTC-3′ for eGFP; the primer locations in the vector are shown in Fig. 1. PCR products were analyzed by agarose gel electrophoresis. The copy number of the transgene was determined by real-time PCR for the eGFP sequence. After the sample was denatured at 95 °C for 2 min, the reaction was started at 94 °C for 5 s, 58 °C for 10 s, and 72 °C for 10 s, for 45 cycles. The hatched chimeric (G0) chickens were mated with wild-type chickens after sexual maturation. The embryonic tissues were collected from fertilized eggs after 6 days of incubation, and the genomic DNA was analyzed.
FIG. 1. Structure of a lentiviral vector pLSi/ΔAeGFP. The vector is indicated in the form of integrated DNA. eGFP, enhanced green fluorescent protein gene; LTR, deleted long terminal repeat; Pact, chicken β-actin promoter; ψ,virus packaging signal sequence; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. Arrows indicate the primers used for the transgene detection. Restriction enzyme sites and locations of probes for Southern blotting are also indicated.
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Analysis of G1 transgenic chickens Genomic DNA extracted from the blood (hatched progeny) or the embryonic body (6-day incubation) was digested with SacI or EcoRI. The digested DNA was electrophoresed on a 0.8% agarose gel and then transferred to a nylon membrane (Hybond-XL; Amersham Biosciences, Piscataway, NJ, USA). The membranes were hybridized with 32P-labeled eGFP and WPRE probes (Fig. 1) prepared from the pLSi/ΔAeGFP plasmid by digestion with NheI plus ClaI and ClaI, respectively, in PerfectHyb Plus Hybridization Buffer (Sigma-Aldrich). To detect expression of the eGFP gene, the heparinized blood sample or embryonic body of the transgenic chicken was analyzed using FACS or observed under a microscope (BZ-8000; Keyence, Osaka, Japan).
RESULTS Purification of PGCs Chicken PGCs have been purified by several methods including Ficoll density gradient centrifugation followed by picking up with a fine needle under a microscope, magnetic cell sorting (MACS), and FACS (16, 17, 26, 27). In the case of MACS and FACS, anti-SSEA-1 or other specific antibodies have been used (16, 27). In this study, PGCs were purified as SSEA-1-positive cells by FACS, which gave high cell purity. In the blood of 2.5-day embryos (Fig. 2A) or in the gonads of 5.5-day embryos (Fig. 2B), the percentages of SSEA-1-positive cells were very low, 0.03% of blood and 1.9% of gonadal cells, respectively. After cell sorting, the purity was 96% and 98% for cPGCs and gPGCs, respectively, which were higher than those obtained by MACS (16 and data not shown). We confirmed that more than 95% of cPGCs and gPGCs were positively stained with anti-VASA antibody (28), which has been widely used as a marker for germ cells (29). We routinely obtained approximately 3000 cPGCs from 30 embryos on day 2.5 and 15,000 gPGCs from 20 embryos on day 5.5. In vitro infection of PGCs cPGCs proliferated poorly in vitro. Therefore, we used an HIV-based replication-defective lentiviral vector, since it can infect and integrate target genes into the genome
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of non-dividing cells (30). To evaluate the infectivity of the lentiviral vector to cPGCs and gPGCs, purified cells were infected with the vector and cultured for 2 days in vitro. During this period, cell number did not increase (data not shown). FACS analysis showed that approximately 30% of cells expressed eGFP for both cPGCs and gPGCs when the cells were infected at MOI 73 (Fig. 3). At present, we cannot estimate the number of PGCs that contain an integrated form of transgene from these data. Migration of both cPGCs and gPGCs into the gonads of recipients Although it was reported that transferred gPGCs can migrate to the gonads in the recipient (16, 19), as well as cPGCs (17, 18), the stage of embryos from which donor PGCs are collected may affect the migration efficiency; this has not been directly compared. To evaluate this, fertilized eggs from a transgenic line harboring a retroviral vector containing GFP and scFvFc genes (7) were used as the source of donor PGCs, which were transferred into the bloodstream of the recipient embryos on day 2.5 (stages 13–16). Recipient embryos were incubated for an additional 7 days (until stage 35), and SSEA-1-positive cells were then purified from the recipients' gonads, and the percentage of donor-derived cells was estimated from the copy number of the retroviral vector. When a small number of cPGCs (∼ 100 cells) was transferred, the cPGCs migrated to the gonads of recipients, and the percentage of the contribution to whole gPGCs was an average of 4% (Fig. 4). Donor gPGCs from 5.5-day embryos also migrated to recipients' gonads despite the mismatch of the developmental stages of donor PGCs and recipient embryos. However, the efficiency was less than half of cPGC (1.5% on average). An increased number of PGCs was then transferred into recipient embryos, and the efficiency of migration was examined (Fig. 4). When 200–250 cPGCs were transferred per embryo, the percentage of migrated cPGCs varied
FIG. 2. Purification of circulating or gonadal PGCs with FACS. Blood cells (A) and gonadal cells (B) were harvested from embryos at stages 13–16 and 27–28, respectively, and were indirectly stained with anti-SSEA-1 antibody. Left, before purification; right, after purification. The percentages of cells in sorted regions (shown as squares) are shown.
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FIG. 3. In vitro infection of circulating or gonadal PGCs by the lentiviral vector. Purified cPGCs (A) and gPGCs (B) were infected with the lentiviral vector (MOI 73) and were cultured for 2 days. The expression of eGFP was examined by FACS. Left, without infection (background control); right, infected with pLSi/ΔAeGFP. The percentages of cells in the region “a” are shown. (C) Summary of three independent cultures. ⁎, p b 0.05 versus non-infected control by Student's t-test. The difference between cPGCs and gPGCs was not statistically significant. The percentage of cells expressing eGFP was 20–40% within a range of 30–100 of MOI (data not shown).
depending on the individual. In the experiments in which more than 300 PGCs were transferred, approximately 10% of gPGCs in the recipient embryos were donor-derived cells, irrespective of the developmental stage of the PGCs, and the efficiency was not obviously increased even when 800–1000 gPGCs were transferred. Overall, the number of PGC delivered to the gonads was not dramatically different between cPGC and gPGC when more than 300 PGCs were transferred per embryo. Production of transgenic chickens with a lentiviral vector The infected gPGCs were transferred into recipient embryos, which were allowed to develop and hatch. The hatchability was 30–50% (data not shown). The value was a little lower than our previous data for the injection of a retroviral vector into the embryonic heart (7). After sexual maturation, the transgene was detected in the genome of sperm from all males (Fig. 5A), and real-time PCR analysis revealed that male #209 had the highest copy number of 0.05 (Fig. 5B). By test crosses between these chimeric chickens, a transgenic progeny could be obtained (male #209 versus female #128, one of 21 embryos
examined) (Fig. 6A). Male #209 was then mated with wild-type female chickens, but no transgenic bird was found in more than 100 offspring analyzed (Table 1). In G0 female chimeras #205 and #225, transgenic offspring were obtained after screening only 30 embryos. Chromosomal DNAs were isolated from three transgenic progeny and were digested with SacI (three sites exist in the transgene) and EcoRI (single site exists in the gene) followed by Southern blotting with two different probes. Southern blotting analysis confirmed that the integrated vector sequence of expected size was detected (4.2 kb; SacI digestion in Fig. 6B). Single site insertion of vector DNA was confirmed in these transgenic chickens since single EcoRI fragments were detected with both probes (Fig. 6B). However, the integration site was different between #205- and #225-derived progeny. Two different probes (WPRE and eGFP) showed bands of the same size for two progeny from #205 (Fig. 6B), suggesting that the integration site of the transgene was the same. The expression of eGFP driven by an actin promoter was examined by FACS in the transgenic progeny G1 #1 from #205. In the blood, a
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FIG. 4. Both cPGC and gPGC migrated into the gonads of recipients. FACS-sorted cPGCs or gPGCs from transgenic chickens were injected into the blood vessel of recipients at stages 13–16. The gonads of the 9-day recipient embryos were collected, and the percentage of donor-derived PGCs was calculated based on the copy number of retroviral genes integrated in donor PGCs. Bars indicate arithmetic means. Open circles and triangles indicate the individual analyses of PGCs from female and male recipients, respectively. Closed squares indicate the analyses of pooled PGC samples from recipients. ⁎, p b 0.05 versus same number of cPGCs by Student's t-test. ⁎⁎, p b 0.05 versus 100 gPGCs by Student's t-test.
small percentage of cells (approximately 2–3%) exhibited strong eGFP expression (Fig. 6C). Microscopic observation revealed that erythrocytes did not express eGFP, possibly reflecting very weak activity of the actin promoter in these cells (Ando, unpublished data), while other cells, such as lymphocytes, expressed eGFP (Fig. 6D). When the embryonic body (G1 #2 from #205) was observed under a microscope, eGFP fluorescence was clearly detected (Fig. 6E). Overall, these results suggest that the G1 offspring expressed the transgene. DISCUSSION Until now, migration of PGCs transferred into the same strain of chickens has not been quantified since a suitable and an easily detectable marker distinguishing donor- and recipient-derived PGCs has not been available. We measured the migration efficiency using transgenic chickens, with the aid of their transgene as a marker. We compared the efficiency of transferred PGCs in migrating to the
FIG. 5. Chimerism of transgenic G0 male. (A) PCR analysis of G0 chicken sperm DNA. The genomic DNA isolated from three G0 male chickens was subjected to PCR. N, genomic DNA isolated from a non-transgenic chicken served as negative control; P, chicken genomic DNA infected by retroviral vector including eGFP sequence as positive control (approximately one copy per cell); MW, DNA size marker. (B) Determination of transgene copy number in the sperm of three G0 male chickens. The genomic DNA in the sperm was analyzed by real-time PCR.
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recipients' gonads between circulating and gonadal PGCs isolated from 2.5- and 5.5-day embryos, respectively. When 100 PGCs were transferred, the percentage of donor-derived PGCs in the recipients' gonads was higher for cPGCs than that for gPGCs. In our protocol, the purified gPGCs were transferred to the embryos that were 2–3 days younger. One possible mechanism for the reduced migration activity of gPGCs is the change in expression of proteins that are essential for circulation and attachment of PGCs to the gonads. In fact, the expression of a chemokine receptor CXCR4, essential for the attachment of cPGC to gonads (31), gradually decreased during PGC development (28). However, if large quantities of gPGCs were used, the migration efficiency was somewhat restored. In fact, we found that the efficiency was comparable when more than 300 PGCs were transferred into recipient embryos. Nevertheless, the migration efficiencies of cPGC and gPGC under optimal conditions were around 10%. Under these conditions, an increased number of PGCs, compared to endogenous PGCs, were transferred. Thus, it is reasonable to assume that the majority of donor PGCs were excluded from the natural delivery process to the gonads. The reason for this is unclear, but it is possible that damage of donor PGCs during manipulation may reduce the efficiency. The number of cPGCs per embryo is in the range of 200–300, but there are 1000 gPGCs per embryo. It is technically difficult and laborious to isolate and purify large quantities of cPGC. Thus, our finding that gPGCs were effectively delivered to the gonads facilitated our trial to establish transgenic chickens using gPGCs. The use of a lentiviral vector, which can deliver and integrate the transgene into the host genome of non-dividing cells (30), is important in enhancing the efficiency of producing transgenic chickens, since the number of chicken PGCs in the M-phase of cell cycle is very low at stages 13–16 (circulating stages) (32). Classical retroviral vectors, such as those based on Moloney murine leukemia virus or murine stem cell virus (MSCV), are not integrated into the chromosome if the cells do not go through M-phase, since the viral cDNA complexed with cellular and viral proteins (preintegration complex) does not have the machinery for nuclear entry (33). Therefore, it is very difficult to introduce transgenes into the genome of PGCs by such vectors. In fact, we observed almost no eGFP expression after 2-day in vitro culture when PGCs were infected with an MSCV-based vector under conditions similar to those in Fig. 3 (data not shown). Furthermore, mating analysis of mature chimeras that received MSCV-infected PGCs showed lower efficiency of germline transmission compared to those produced using an HIV-based vector (data not shown). The copy number of transgenes in sperm was generally low in our experiments (below 0.05; Fig. 5), and no transgenic chickens were produced by mating the male G0 chicken with the highest copy number (#209) with wild-type females, even after analyzing a theoretically large number of offspring. However, transgenic offspring could be obtained at a frequency of around 3–7% from G0 female chickens (Table 1). Judging from the efficiency of lentiviral infection to PGCs (approximately 30%) and that of PGCs delivery to the gonads (10%), this frequency of establishing transgenic chickens seems to be reasonable. This frequency could be acceptable, but there are several possibilities to further improve the efficiency. One is to reduce the possible damages of donor PGCs by, for example, the use of alternative methods such as density gradient centrifugation with Ficoll (26) or Nycodenz (34). The other is extensive depletion of endogenous PGCs. For this, several strategies may be available. For instance, we will be able to remove the increasing amount of blood without severe reduction of the viability of embryos (17). Sterilization of endogenous PGCs with ultraviolet (35) or γ-ray (36) may be the alternative choice. Until now, transgenic progeny have been screened from G0 male chickens in almost all studies (7-9, 12, 13), since more chicks can be
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FIG. 6. Analysis of transgenic G1 chicken produced with lentiviral vector. (A) Representative results of mating. Progeny of #128 mated with #209 are shown. Embryo #3 was confirmed to be transgenic G1 progeny. N, wild-type genomic DNA as negative control; P, plasmid DNA as positive control. (B) Southern blotting analysis of blood (#205-1) or embryonic bodies (#205-2 and #225-1) of transgenic chickens. Two transgenic progeny (#1 and #2) were from #205 female, and one from #225 female. (C, D) eGFP expression in the blood of a G1 transgenic chicken (#205-1). (C) Blood cells of either non-transgenic (WT, left) or transgenic (right) chickens were analyzed by FACS. (D) Blood cells of transgenic chicken (#205-1) were observed using a fluorescent microscope. Left, fluorescence image; right, phase contrast. Small blood cells (indicated by arrowheads), but not erythrocytes (indicated by asterisks), expressed eGFP. Scale bars indicate 10 μm. (E) eGFP expression in a piece of the embryonic body of G1 transgenic chicken (#205-2). Left, non-transgenic chicken; right, transgenic chicken. Scale bars indicate 200 μm.
obtained by artificial insemination compared with G0 female chickens. However, our study suggests that female chickens can be a good source of G1 progeny. We observed that the percentage of donorderived PGCs tended to be higher in female recipients than in male recipients when recipient embryos were individually examined (Fig. 4). Naito et al. reported that the percentage of progeny derived from donor PGCs was dependent on gender (17, 18). We need further investigation to clarify the reason why male chimeras show lower efficiency of transmission than females.
According to Southern blotting, it appears that the integration sites of the transgene in the two progeny from #205 female were the same. Since integration of HIV and HIV-based vectors in chromosome occurs at random, except that they favor actively transcribed regions (37), it is unlikely that these two transgenic chickens are derived from two different PGCs in which independent integration events occurred in the same site. Instead, these two transgenic offspring probably derived from ova differentiated from the same PGC by cell division, suggesting that a minimal number of
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TABLE 1. List of representative G0 chickens with lentivirus-infected gPGCs. No. #128 #205 #209 #225 #226 #511 #513 a
Sex a
gPGCs injected per embryo
MOI
Number of progeny examined
G1 (%)
F F M F F F F
1600 1200
55 54
850
77
1500
24
36 30 101 30 33 34 32
0 2 (6.66) 0 1 (3.33) 0 0 0
F, female; M, male.
gPGCs may develop to matured ova. Overall, we could obtain transgenic chickens using gPGCs and a lentiviral vector with a substantially acceptable frequency. ACKNOWLEDGMENTS We thank the Radio Isotope Research Center of Nagoya University for supporting the radioisotope experiments, and Co60 radiation facility of Nagoya University for sterilization of host eggshells. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) by Bio-oriented Technology Research Advancement Institution (BRAIN). References 1. Houdebine, L. M.: Transgenic animal bioreactors, Transgenic Res., 9, 305–320 (2000). 2. Ivarie, R.: Avian transgenesis: progress towards the promise, Trends Biotechnol., 21, 14–19 (2003). 3. Raju, T. S., Briggs, J. B., Borge, S. M., and Jones, A. J.: Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branchspecific galactosylation and importance for engineering recombinant glycoprotein therapeutics, Glycobiology, 10, 477–486 (2000). 4. Salter, D. W., Smith, E. J., Hughes, S. H., Wright, S. E., and Crittenden, L. B.: Transgenic chickens: insertion of retroviral genes into the chicken germ line, Virology, 157, 236–240 (1987). 5. Bosselman, R. A., Hsu, R. Y., Boggs, T., Hu, S., Bruszewski, J., Ou, S., Kozar, L., Martin, F., Green, C., and Jacobsen, F., et al.: Germline transmission of exogenous genes in the chicken, Science, 243, 533–535 (1989). 6. Kwon, M. S., Koo, B. C., Choi, B. R., Lee, H. T., Kim, Y. H., Ryu, W. S., Shim, H., Kim, J. H., Kim, N. H., and Kim, T.: Development of transgenic chickens expressing enhanced green fluorescent protein, Biochem. Biophys. Res. Commun., 320, 442–448 (2004). 7. Kamihira, M., Ono, K., Esaka, K., Nishijima, K., Kigaku, R., Komatsu, H., Yamashita, T., Kyogoku, K., and Iijima, S.: High-level expression of single-chain Fv-Fc fusion protein in serum and egg white of genetically manipulated chickens by using a retroviral vector, J. Virol., 79, 10864–10874 (2005). 8. Harvey, A. J., Speksnijder, G., Baugh, L. R., Morris, J. A., and Ivarie, R.: Expression of exogenous protein in the egg white of transgenic chickens, Nat. Biotechnol., 20, 396–399 (2002). 9. Rapp, J. C., Harvey, A. J., Speksnijder, G. L., Hu, W., and Ivarie, R.: Biologically active human interferon α-2b produced in the egg white of transgenic hens, Transgenic Res., 12, 569–575 (2003). 10. Kodama, D., Nishimiya, D., Iwata, K., Yamaguchi, K., Yoshida, K., Kawabe, Y., Motono, M., Watanabe, H., Yamashita, T., Nishijima, K., Kamihira, M., and Iijima, S.: Production of human erythropoietin by chimeric chickens, Biochem. Biophys. Res. Commun., 367, 834–839 (2008). 11. Kyogoku, K., Yoshida, K., Watanabe, H., Yamashita, T., Kawabe, Y., Motono, M., Nishijima, K., Kamihira, M., and Iijima, S.: Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens, J. Biosci. Bioeng., 105, 454–459 (2008). 12. Lillico, S. G., Sherman, A., McGrew, M. J., Robertson, C. D., Smith, J., Haslam, C., Barnard, P., Radcliffe, P. A., Mitrophanous, K. A., Elliot, E. A., and Sang, H. M.: Oviduct-specific expression of two therapeutic proteins in transgenic hens, Proc. Natl. Acad. Sci. U. S. A., 104, 1771–1776 (2007).
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13. McGrew, M. J., Sherman, A., Ellard, F. M., Lillico, S. G., Gilhooley, H. J., Kingsman, A. J., Mitrophanous, K. A., and Sang, H.: Efficient production of germline transgenic chickens using lentiviral vectors, EMBO Rep., 5, 728–733 (2004). 14. Chapman, S. C., Lawson, A., Macarthur, W. C., Wiese, R. J., Loechel, R. H., Burgos-Trinidad, M., Wakefield, J. K., Ramabhadran, R., Mauch, T. J., and Schoenwolf, G. C.: Ubiquitous GFP expression in transgenic chickens using a lentiviral vector, Development, 132, 935–940 (2005). 15. D'Costa, S., Pardue, S. L., and Petitte, J. N.: Comparative development of avian primordial germ cells and production of germ line chimeras, Avian Poult. Biol. Rev., 12, 151–168 (2001). 16. Kim, J. N., Kim, M. A., Park, T. S., Kim, D. K., Park, H. J., Ono, T., Lim, J. M., and Han, J. Y.: Enriched gonadal migration of donor-derived gonadal primordial germ cells by immunomagnetic cell sorting in birds, Mol. Reprod. Dev., 68, 81–87 (2004). 17. Naito, M., Tajima, A., Yasuda, Y., and Kuwana, T.: Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cells, Mol. Reprod. Dev., 39, 153–161 (1994). 18. Naito, M., Matsubara, Y., Harumi, T., Tagami, T., Kagami, H., Sakurai, M., and Kuwana, T.: Differentiation of donor primordial germ cells into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells isolated from embryonic blood, J. Reprod. Fertil., 117, 291–298 (1999). 19. Mozdziak, P. E., Wysocki, R., Angerman-Stewart, J., Pardue, S. L., and Petitte, J. N.: Production of chick germline chimeras from fluorescence-activated cellsorted gonocytes, Poult. Sci., 85, 1764–1768 (2006). 20. Vick, L., Li, Y., and Simkiss, K.: Transgenic birds from transformed primordial germ cells, Proc. Biol. Sci., 251, 179–182 (1993). 21. Shin, S. S., Kim, T. M., Kim, S. Y., Kim, T. W., Seo, H. W., Lee, S. K., Kwon, S. C., Lee, G. S., Kim, H., Lim, J. M., and Han, J. Y.: Generation of transgenic quail through germ cell-mediated germline transmission, FASEB J., 22, 2435–2444 (2008). 22. van de Lavoir, M. C., Diamond, J. H., Leighton, P. A., Mather-Love, C., Heyer, B. S., Bradshaw, R., Kerchner, A., Hooi, L. T., Gessaro, T. M., Swanberg, S. E., Delany, M. E., and Etches, R. J.: Germline transmission of genetically modified primordial germ cells, Nature, 441, 766–769 (2006). 23. Hamburger, V. and Hamilton, H. L.: A series of normal stages in the development of the chick embryo, J. Morphol., 88, 49–92 (1951). 24. Ventura, A., Meissner, A., Dillon, C. P., McManus, M., Sharp, P. A., Van Parijs, L., Jaenisch, R., and Jacks, T.: Cre-lox-regulated conditional RNA interference from transgenes, Proc. Natl. Acad. Sci. U. S. A., 101, 10380–10385 (2004). 25. Suemori, H., Kadodawa, Y., Goto, K., Araki, I., Kondoh, H., and Nakatsuji, N.: A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous β-galactosidase expression, Cell Differ. Dev., 29, 181–186 (1990). 26. Yasuda, Y., Tajima, A., Fujimoto, T., and Kuwana, T.: A method to obtain avian germ-line chimaeras using isolated primordial germ cells, J. Reprod. Fertil., 96, 521–528 (1992). 27. Mozdziak, P. E., Angerman-Stewart, J., Rushton, B., Pardue, S. L., and Petitte, J. N.: Isolation of chicken primordial germ cells using fluorescence-activated cell sorting, Poult. Sci., 84, 594–600 (2005). 28. Motono, M., Ohashi, T., Nishijima, K., and Iijima, S.: Analysis of chicken primordial germ cells, Cytotechnology, 57, 199–205 (2008). 29. Tsunekawa, N., Naito, M., Sakai, Y., Nishida, T., and Noce, T.: Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells, Development, 127, 2741–2750 (2000). 30. Suzuki, Y. and Craigie, R.: The road to chromatin - nuclear entry of retroviruses, Nat. Rev. Microbiol., 5, 187–196 (2007). 31. Stebler, J., Spieler, D., Slanchev, K., Molyneaux, K. A., Richter, U., Cojocaru, V., Tarabykin, V., Wylie, C., Kessel, M., and Raz, E.: Primordial germ cell migration in the chick and mouse embryo: the role of the chemokine SDF-1/CXCL12, Dev. Biol., 272, 351–361 (2004). 32. Maeda, T.: Analysis of the mitotic index of chicken primordial germ cells before and after settling in the germinal ridge, J. Reprod. Fertil., 113, 211–215 (1998). 33. Roe, T., Reynolds, T. C., Yu, G., and Brown, P. O.: Integration of murine leukemia virus DNA depends on mitosis, EMBO J., 12, 2099–2108 (1993). 34. Zhao, D. F. and Kuwana, T.: Purification of avian circulating primordial germ cells by Nycodenz density gradient centrifugation, Br. Poult. Sci., 44, 30–35 (2003). 35. Aige-Gil, V. and Simkiss, K.: Sterilising embryos for transgenic chimaeras, Br. Poult. Sci., 32, 427–438 (1991). 36. Carsience, R. S., Clark, M. E., Verrinder Gibbins, A. M., and Etches, R. J.: Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos, Development, 117, 669–675 (1993). 37. Ellis, J.: Silencing and variegation of gammaretrovirus and lentivirus vectors, Hum. Gene Ther., 16, 1241–1246 (2005).
Production of transgenic chickens from purified primordial germ cells infected with a lentiviral vector Makoto Motono, Yuki Yamada, Yuki Hattori, Ryo Nakagawa, Ken-ichi Nishijima,⁎ and Shinji Iijima Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan Received 19 August 2009; accepted 6 October 2009 Available online 29 October 2009
Replication-defective retroviral or lentiviral vectors have been used for the production of transgenic animals. Chicken primordial germ cells (PGCs) are the precursors for ova and spermatozoa. Here, we describe the production of transgenic chickens via a germline transmission system using PGCs infected with a replication-defective lentiviral vector. PGCs were sorted with a fluorescence-activated cell sorter based on the expression of stage-specific embryonic antigen-1 from 2.5- and 5.5-day embryos. PGCs from both stages of embryo were infected with a lentiviral vector at a similar efficiency in vitro. PGCs were then transferred into the bloodstream of 2.5-day recipient embryos. The efficiency with which the PGCs were delivered and settled in the gonads was lower for PGCs from 5.5-day embryos than those from 2.5-day embryos when a limited number of PGCs was transferred, while the difference was not obvious upon the transfer of increased number of cells. Using a high number of 5.5-day PGCs infected with a lentiviral vector, transgenic chimeras (G0) with an acceptable efficiency for germline transmission were obtained. G0 female chickens produced transgenic progeny (G1) with higher efficiency compared to G0 male chickens. In G1 transgenic chickens obtained by this method, enhanced green fluorescent protein was effectively expressed under the control of the actin promoter. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Transgenic chicken; Transgenic avian bioreactor; Lentiviral vector; Primordial germ cell; Cell sorter]
Transgenic animals have been generated for the production of recombinant proteins in the milk of livestock species such as goats, sheep, pigs, and cows (1). However, mammalian transgenic systems have several drawbacks in that they require a relatively large area for breeding and a few years to become sexually mature. Therefore, avian species have attracted great attention as an alternative choice (2). In particular, chickens have several advantages such as high protein productivity in eggs, the small space required for breeding, and the lack of prion diseases. In addition, the N-glycosylated carbohydrate of chicken has N-acetylneuraminic acid at its end as in that of human (3). Several researchers have produced transgenic chickens by injecting viral vectors into embryos (4-14). Retroviral vectors derived from avian leukosis virus, reticuloendotheliosis virus, or Moloney murine leukemia virus have been injected into the subgerminal cavity at the blastodermal stage or into the heart of developing embryos to obtain transgenic chickens (4-11). Recently, lentiviral vectors have been used to produce transgenic chickens since they are relatively resistant to gene silencing (12-14). We and other research groups have reported on the production of recombinant proteins into eggs using genetically manipulated chickens (7-12), but in all cases, germline transmission efficiency is relatively low, which must be overcome for the practical application of transgenic technology in chickens.
⁎ Corresponding author. Tel.: +81 52 789 4279; fax: +81 52 789 3221. E-mail address: [email protected] (K. Nishijima).
Primordial germ cells (PGCs) are progenitor cells of ova and spermatozoa. Chicken PGCs can be recognized at the center of the area pellucida in the stage X blastoderm (freshly laid eggs). After gastrulation, they can be found in the extraembryonic region, the germinal crescent. They then enter and circulate in the blood vessels of the embryo, and migrate to the gonadal ridges, which develop into the mature gonads (15). Previous experiments showed that isolated PGCs could be transferred to other recipient embryos at the stages in which PGCs circulate in the blood, without losing the ability to contribute to the formation of germ cells (16-19). Thus, it is assumed that gene transfer to PGCs and subsequent transplantation to recipient embryos is an alternative method to generate transgenic chickens. Furthermore, it is expected that the use of PGC may improve the efficiency of germline transmission since PGCs are progenitors of ova and spermatozoa. In the early 1990s, it was shown that transgenic chickens could be established with PGCs infected with spleen necrosis virus, but a detailed analysis was not reported (20). Last year, transgenic quails were established using PGCs, but the efficiency was low (21). In addition, transgenic chickens could be obtained by transferring PGCs that had been cultured for more than one month, which permitted the selection of cells containing an integrated form of transgene (22). However, long-term cultivation of PGCs is difficult, and the efficiency of obtaining transgenic progeny varies across research groups possibly due to different experimental methods. One important factor determining the efficiency of obtaining transgenic progeny is the percentage of the transferred PGCs to the
1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.10.007
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whole PGC population (transferred plus endogenous). Experiments in which PGCs were transferred without gene manipulation showed that the percentages of progeny from donor-derived PGCs varied depending on various experimental factors including the strain of chicken, source of PGCs, methods for PGC purification, and sex of birds (15-19). In these experiments, circulating PGCs (cPGCs) obtained from 2.5-day embryos were transferred to the blood vessels of the recipient embryos in the same developmental stage (17, 18). In addition, gonadal PGCs (gPGCs) from 5- to 5.5-day embryos were also used to produce germline chimeras despite the mismatch of their developmental stages (16, 19). However, direct comparison of the efficiency with which PGCs are delivered to the gonads of recipients has not been performed for cPGC and gPGC thus far. In order to optimize conditions for the establishment of transgenic chickens, we directly compared the efficiency of PGCs from 2.5- and 5.5-day embryos in migrating to the recipients' gonads and succeeded in establishing transgenic progeny using gPGCs infected with a lentiviral vector. MATERIALS AND METHODS Preparation of fertilized eggs Fertilized eggs were obtained by artificial insemination of White Leghorn (line M; originally obtained from Nisseiken, Yamanashi, Japan). The laid eggs were sterilized with 70% ethanol and were preserved at 12 °C until use. All experiments were performed according to the ethical guidelines of Nagoya University for animal experimentation. Preparation of cPGCs and gPGCs Fertilized eggs were incubated at 38 °C and 65% humidity with rocking every 15 min at an angle of 90°. To obtain blood cells, the sharp end of the eggs was cut horizontally after incubation for 51 h. After confirming that the embryos had developed to Hamburger–Hamilton stages 13–16 (23), blood was drawn with a glass micropipette from 30 to 40 embryos and resuspended in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, USA). The cells were centrifuged at 200 × g for 5 min at 4 °C and were washed with phosphate-buffered saline (PBS) containing 0.5% FBS. To obtain gonadal cells, embryos were isolated from fertilized eggs at day 5.5 of incubation (stages 27–28) and rinsed with PBS. Gonads were isolated with forceps and were dispersed in 0.1% trypsin and 0.02% EDTA. DMEM containing 10% FBS was added to stop trypsin digestion, and the cells were washed as described for blood cells. To purify cPGCs and gPGCs, dispersed blood and gonadal cells were incubated with 100 μl of a 1:100 dilution (in PBS containing 0.5% FBS) of anti-stage-specific embryonic antigen (SSEA)-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min on ice. After washing with PBS, 100 μl of a 1:200 dilution (in PBS containing 0.5% FBS) of phycoerythrin-labeled goat anti-mouse IgM antibody (Rockland, Gilbertsville, PA, USA) was added to the cell suspension and incubated for 30 min on ice. The suspension was washed with PBS and resuspended in 1 ml of PBS containing 0.5% FBS. Cells expressing SSEA-1 were sorted using a fluorescence-activated cell sorter (FACS) (EPICS ALTRA; Beckman-Coulter, Fullerton, CA, USA). The sorted SSEA-1-positive cells were resuspended in DMEM containing 10% FBS at a cell concentration of 100–200 cPGCs/ μl or 300–500 gPGCs/μl, based on cell counting by FACS. Plasmid construction Human immunodeficiency virus (HIV)-based lentiviral vector plasmid pSicoR (Addgene plasmid 11579) (24) was obtained from Addgene (Cambridge, MA, USA). It was digested with XbaI and NheI to remove the cytomegalovirus promoter, and the chicken β-actin promoter derived from pmiwZ (25) was inserted into the same restriction site to construct pLSi/ΔAeGFP, in which enhanced green fluorescent protein (eGFP) was expressed from the β-actin promoter (Fig. 1). Lentiviral vector production 293FT cells seeded on 24-well collagen-coated plates (Asahi Techno Glass, Chiba, Japan) were transfected with 300 ng of lentiviral
J. BIOSCI. BIOENG., vector (pLSi/ΔAeGFP), 100 ng of gag/pol plasmid (pLP1), 240 ng of rev plasmid (pLP2), and 160 ng of VSV-G plasmid (pLP VSV-G) (Invitrogen, Carlsbad, CA, USA) using Lipofectamine 2000 (Invitrogen). At 48 h posttransfection, the culture supernatant was collected and filtered through a 0.45-μm filter, followed by centrifugation at 20,000 × g for 5 h at 4 °C. The viral pellet was suspended in 7–10 μl of 50 mM Tris-HCl buffer (pH 7.8) containing 130 mM NaCl and 1 mM EDTA. Polybrene (Sigma-Aldrich) was added to the solution at a concentration of 8 μg/ml before the infection. The viral titer was determined with HeLa cells in terms of the percentage of cells expressing the eGFP gene. Viral infection in cultured PGCs The cPGCs and gPGCs were incubated with the lentiviral vector at a multiplicity of infection (MOI) of 30–100 in DMEM containing 10 % FBS for 2–4 h at 37 °C. The cells were then cultured on 96-well gelatin-coated microplates (Asahi Techno Glass) at 37 °C for 2 days in DMEM supplemented with 10% FBS, 10 mM HEPES (Nakarai Tesque, Kyoto, Japan), 1 × non-essential amino acids (Invitrogen), 10 ng/ml insulin-like growth factor-I (Invitrogen), 10 ng/ml basic fibroblast growth factor (Invitrogen), and 10 units/ml murine leukemia inhibitory factor (ESGRO; Chemicon International, Temecula, CA, USA) and were analyzed using FACS for the expression of eGFP. Analysis of transferred PGCs in the gonads of 9-day recipients To assess the percentage of donor-derived PGCs that had migrated and settled in the gonads of the recipients, PGCs derived from transgenic chickens producing single-chain antibody, established previously in our laboratory (7), were used as donor cells. Sorted PGCs were injected into the blood vessel of stages 13–16 recipient embryo. The sharp end of fertilized eggs that had been incubated for 51 h was cut horizontally to make an opening of 35 mm in diameter. To reduce the number of endogenous cPGCs in each recipient, the blood vessel of stages 13–16 recipient embryo was pricked with a fine glass micropipette, and 2–4 μl of blood was bled out as described previously (17). The sorted cells were then injected into the blood vessel of each manipulated embryo followed by insertion of bubbles to stop bleeding from the point of injection (26). The embryos were developed in surrogate chicken eggshells that had been sterilized by γradiation from 60Co prior to use. After incubation for 7 days, the recipient embryos were washed with PBS, and the gonads were dissected. The SSEA-1-positive cells were sorted by FACS and were preserved at -80 °C until analysis. In some experiments, the sex of recipient embryos was determined by morphological differences of the gonads, and each embryo was separately subjected to cell sorting. Genomic DNA was extracted from the sorted cells using a DNA preparation kit (QIAamp DNA Micro Kit; Qiagen, Hilden, Germany) according to the manufacturer's instructions. The percentage of donorderived cells that possessed the transgene was determined by real-time polymerase chain reaction (PCR) (LightCycler 1.5 Instrument; Roche Diagnostics, Mannheim, Germany) to amplify the retroviral vector sequence (packaging signal) using hybridization probes as described previously (7). The amount of genomic DNA was normalized by real-time PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH was amplified in 20 μl reaction mixture containing 10 μl Platinum SYBRGreen qPCR SuperMix-UDG (Invitrogen), 500 nM each primer, 1 μl 20 × bovine serum albumin (1 mg/ml), and 2 μl sample DNA with 45 cycles (94 °C for 5 s, 55 °C for 10 s, and 72 °C 10 s). The primers used were 5′-GGGCACGCCATCACTATC-3′ and 5′-GTGAAGACACCAGTGGACTCC-3′. Production of germline chimeric chickens The sorted gPGCs were incubated with the lentiviral vector at various MOI in DMEM containing 10% FBS for 2–4 h at 37 °C. To produce transgenic chimeras, the cells were injected into the blood vessels of stages 13–16 recipient embryos. One day before the expected day of hatching, rocking was stopped, and the plastic film was punctured to assist embryonic pulmonary respiration. Transgene detection by PCR Genomic DNA from the sperm was extracted using MagExtractor genome (Toyobo, Osaka, Japan). PCR was performed with 20 ng of the genomic DNA as a template. After initial denaturation of DNA at 94 °C for 1 min, PCR was started at 94 °C for 15 s, 58 °C for 30 s, and 68 °C for 15 s, for 35 cycles. The primers used were 5′-CGGCAACTACAAGACCCGC-3′ and 5′-GAAGTTCACCTTGATGCCGTTC-3′ for eGFP; the primer locations in the vector are shown in Fig. 1. PCR products were analyzed by agarose gel electrophoresis. The copy number of the transgene was determined by real-time PCR for the eGFP sequence. After the sample was denatured at 95 °C for 2 min, the reaction was started at 94 °C for 5 s, 58 °C for 10 s, and 72 °C for 10 s, for 45 cycles. The hatched chimeric (G0) chickens were mated with wild-type chickens after sexual maturation. The embryonic tissues were collected from fertilized eggs after 6 days of incubation, and the genomic DNA was analyzed.
FIG. 1. Structure of a lentiviral vector pLSi/ΔAeGFP. The vector is indicated in the form of integrated DNA. eGFP, enhanced green fluorescent protein gene; LTR, deleted long terminal repeat; Pact, chicken β-actin promoter; ψ,virus packaging signal sequence; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. Arrows indicate the primers used for the transgene detection. Restriction enzyme sites and locations of probes for Southern blotting are also indicated.
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Analysis of G1 transgenic chickens Genomic DNA extracted from the blood (hatched progeny) or the embryonic body (6-day incubation) was digested with SacI or EcoRI. The digested DNA was electrophoresed on a 0.8% agarose gel and then transferred to a nylon membrane (Hybond-XL; Amersham Biosciences, Piscataway, NJ, USA). The membranes were hybridized with 32P-labeled eGFP and WPRE probes (Fig. 1) prepared from the pLSi/ΔAeGFP plasmid by digestion with NheI plus ClaI and ClaI, respectively, in PerfectHyb Plus Hybridization Buffer (Sigma-Aldrich). To detect expression of the eGFP gene, the heparinized blood sample or embryonic body of the transgenic chicken was analyzed using FACS or observed under a microscope (BZ-8000; Keyence, Osaka, Japan).
RESULTS Purification of PGCs Chicken PGCs have been purified by several methods including Ficoll density gradient centrifugation followed by picking up with a fine needle under a microscope, magnetic cell sorting (MACS), and FACS (16, 17, 26, 27). In the case of MACS and FACS, anti-SSEA-1 or other specific antibodies have been used (16, 27). In this study, PGCs were purified as SSEA-1-positive cells by FACS, which gave high cell purity. In the blood of 2.5-day embryos (Fig. 2A) or in the gonads of 5.5-day embryos (Fig. 2B), the percentages of SSEA-1-positive cells were very low, 0.03% of blood and 1.9% of gonadal cells, respectively. After cell sorting, the purity was 96% and 98% for cPGCs and gPGCs, respectively, which were higher than those obtained by MACS (16 and data not shown). We confirmed that more than 95% of cPGCs and gPGCs were positively stained with anti-VASA antibody (28), which has been widely used as a marker for germ cells (29). We routinely obtained approximately 3000 cPGCs from 30 embryos on day 2.5 and 15,000 gPGCs from 20 embryos on day 5.5. In vitro infection of PGCs cPGCs proliferated poorly in vitro. Therefore, we used an HIV-based replication-defective lentiviral vector, since it can infect and integrate target genes into the genome
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of non-dividing cells (30). To evaluate the infectivity of the lentiviral vector to cPGCs and gPGCs, purified cells were infected with the vector and cultured for 2 days in vitro. During this period, cell number did not increase (data not shown). FACS analysis showed that approximately 30% of cells expressed eGFP for both cPGCs and gPGCs when the cells were infected at MOI 73 (Fig. 3). At present, we cannot estimate the number of PGCs that contain an integrated form of transgene from these data. Migration of both cPGCs and gPGCs into the gonads of recipients Although it was reported that transferred gPGCs can migrate to the gonads in the recipient (16, 19), as well as cPGCs (17, 18), the stage of embryos from which donor PGCs are collected may affect the migration efficiency; this has not been directly compared. To evaluate this, fertilized eggs from a transgenic line harboring a retroviral vector containing GFP and scFvFc genes (7) were used as the source of donor PGCs, which were transferred into the bloodstream of the recipient embryos on day 2.5 (stages 13–16). Recipient embryos were incubated for an additional 7 days (until stage 35), and SSEA-1-positive cells were then purified from the recipients' gonads, and the percentage of donor-derived cells was estimated from the copy number of the retroviral vector. When a small number of cPGCs (∼ 100 cells) was transferred, the cPGCs migrated to the gonads of recipients, and the percentage of the contribution to whole gPGCs was an average of 4% (Fig. 4). Donor gPGCs from 5.5-day embryos also migrated to recipients' gonads despite the mismatch of the developmental stages of donor PGCs and recipient embryos. However, the efficiency was less than half of cPGC (1.5% on average). An increased number of PGCs was then transferred into recipient embryos, and the efficiency of migration was examined (Fig. 4). When 200–250 cPGCs were transferred per embryo, the percentage of migrated cPGCs varied
FIG. 2. Purification of circulating or gonadal PGCs with FACS. Blood cells (A) and gonadal cells (B) were harvested from embryos at stages 13–16 and 27–28, respectively, and were indirectly stained with anti-SSEA-1 antibody. Left, before purification; right, after purification. The percentages of cells in sorted regions (shown as squares) are shown.
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FIG. 3. In vitro infection of circulating or gonadal PGCs by the lentiviral vector. Purified cPGCs (A) and gPGCs (B) were infected with the lentiviral vector (MOI 73) and were cultured for 2 days. The expression of eGFP was examined by FACS. Left, without infection (background control); right, infected with pLSi/ΔAeGFP. The percentages of cells in the region “a” are shown. (C) Summary of three independent cultures. ⁎, p b 0.05 versus non-infected control by Student's t-test. The difference between cPGCs and gPGCs was not statistically significant. The percentage of cells expressing eGFP was 20–40% within a range of 30–100 of MOI (data not shown).
depending on the individual. In the experiments in which more than 300 PGCs were transferred, approximately 10% of gPGCs in the recipient embryos were donor-derived cells, irrespective of the developmental stage of the PGCs, and the efficiency was not obviously increased even when 800–1000 gPGCs were transferred. Overall, the number of PGC delivered to the gonads was not dramatically different between cPGC and gPGC when more than 300 PGCs were transferred per embryo. Production of transgenic chickens with a lentiviral vector The infected gPGCs were transferred into recipient embryos, which were allowed to develop and hatch. The hatchability was 30–50% (data not shown). The value was a little lower than our previous data for the injection of a retroviral vector into the embryonic heart (7). After sexual maturation, the transgene was detected in the genome of sperm from all males (Fig. 5A), and real-time PCR analysis revealed that male #209 had the highest copy number of 0.05 (Fig. 5B). By test crosses between these chimeric chickens, a transgenic progeny could be obtained (male #209 versus female #128, one of 21 embryos
examined) (Fig. 6A). Male #209 was then mated with wild-type female chickens, but no transgenic bird was found in more than 100 offspring analyzed (Table 1). In G0 female chimeras #205 and #225, transgenic offspring were obtained after screening only 30 embryos. Chromosomal DNAs were isolated from three transgenic progeny and were digested with SacI (three sites exist in the transgene) and EcoRI (single site exists in the gene) followed by Southern blotting with two different probes. Southern blotting analysis confirmed that the integrated vector sequence of expected size was detected (4.2 kb; SacI digestion in Fig. 6B). Single site insertion of vector DNA was confirmed in these transgenic chickens since single EcoRI fragments were detected with both probes (Fig. 6B). However, the integration site was different between #205- and #225-derived progeny. Two different probes (WPRE and eGFP) showed bands of the same size for two progeny from #205 (Fig. 6B), suggesting that the integration site of the transgene was the same. The expression of eGFP driven by an actin promoter was examined by FACS in the transgenic progeny G1 #1 from #205. In the blood, a
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FIG. 4. Both cPGC and gPGC migrated into the gonads of recipients. FACS-sorted cPGCs or gPGCs from transgenic chickens were injected into the blood vessel of recipients at stages 13–16. The gonads of the 9-day recipient embryos were collected, and the percentage of donor-derived PGCs was calculated based on the copy number of retroviral genes integrated in donor PGCs. Bars indicate arithmetic means. Open circles and triangles indicate the individual analyses of PGCs from female and male recipients, respectively. Closed squares indicate the analyses of pooled PGC samples from recipients. ⁎, p b 0.05 versus same number of cPGCs by Student's t-test. ⁎⁎, p b 0.05 versus 100 gPGCs by Student's t-test.
small percentage of cells (approximately 2–3%) exhibited strong eGFP expression (Fig. 6C). Microscopic observation revealed that erythrocytes did not express eGFP, possibly reflecting very weak activity of the actin promoter in these cells (Ando, unpublished data), while other cells, such as lymphocytes, expressed eGFP (Fig. 6D). When the embryonic body (G1 #2 from #205) was observed under a microscope, eGFP fluorescence was clearly detected (Fig. 6E). Overall, these results suggest that the G1 offspring expressed the transgene. DISCUSSION Until now, migration of PGCs transferred into the same strain of chickens has not been quantified since a suitable and an easily detectable marker distinguishing donor- and recipient-derived PGCs has not been available. We measured the migration efficiency using transgenic chickens, with the aid of their transgene as a marker. We compared the efficiency of transferred PGCs in migrating to the
FIG. 5. Chimerism of transgenic G0 male. (A) PCR analysis of G0 chicken sperm DNA. The genomic DNA isolated from three G0 male chickens was subjected to PCR. N, genomic DNA isolated from a non-transgenic chicken served as negative control; P, chicken genomic DNA infected by retroviral vector including eGFP sequence as positive control (approximately one copy per cell); MW, DNA size marker. (B) Determination of transgene copy number in the sperm of three G0 male chickens. The genomic DNA in the sperm was analyzed by real-time PCR.
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recipients' gonads between circulating and gonadal PGCs isolated from 2.5- and 5.5-day embryos, respectively. When 100 PGCs were transferred, the percentage of donor-derived PGCs in the recipients' gonads was higher for cPGCs than that for gPGCs. In our protocol, the purified gPGCs were transferred to the embryos that were 2–3 days younger. One possible mechanism for the reduced migration activity of gPGCs is the change in expression of proteins that are essential for circulation and attachment of PGCs to the gonads. In fact, the expression of a chemokine receptor CXCR4, essential for the attachment of cPGC to gonads (31), gradually decreased during PGC development (28). However, if large quantities of gPGCs were used, the migration efficiency was somewhat restored. In fact, we found that the efficiency was comparable when more than 300 PGCs were transferred into recipient embryos. Nevertheless, the migration efficiencies of cPGC and gPGC under optimal conditions were around 10%. Under these conditions, an increased number of PGCs, compared to endogenous PGCs, were transferred. Thus, it is reasonable to assume that the majority of donor PGCs were excluded from the natural delivery process to the gonads. The reason for this is unclear, but it is possible that damage of donor PGCs during manipulation may reduce the efficiency. The number of cPGCs per embryo is in the range of 200–300, but there are 1000 gPGCs per embryo. It is technically difficult and laborious to isolate and purify large quantities of cPGC. Thus, our finding that gPGCs were effectively delivered to the gonads facilitated our trial to establish transgenic chickens using gPGCs. The use of a lentiviral vector, which can deliver and integrate the transgene into the host genome of non-dividing cells (30), is important in enhancing the efficiency of producing transgenic chickens, since the number of chicken PGCs in the M-phase of cell cycle is very low at stages 13–16 (circulating stages) (32). Classical retroviral vectors, such as those based on Moloney murine leukemia virus or murine stem cell virus (MSCV), are not integrated into the chromosome if the cells do not go through M-phase, since the viral cDNA complexed with cellular and viral proteins (preintegration complex) does not have the machinery for nuclear entry (33). Therefore, it is very difficult to introduce transgenes into the genome of PGCs by such vectors. In fact, we observed almost no eGFP expression after 2-day in vitro culture when PGCs were infected with an MSCV-based vector under conditions similar to those in Fig. 3 (data not shown). Furthermore, mating analysis of mature chimeras that received MSCV-infected PGCs showed lower efficiency of germline transmission compared to those produced using an HIV-based vector (data not shown). The copy number of transgenes in sperm was generally low in our experiments (below 0.05; Fig. 5), and no transgenic chickens were produced by mating the male G0 chicken with the highest copy number (#209) with wild-type females, even after analyzing a theoretically large number of offspring. However, transgenic offspring could be obtained at a frequency of around 3–7% from G0 female chickens (Table 1). Judging from the efficiency of lentiviral infection to PGCs (approximately 30%) and that of PGCs delivery to the gonads (10%), this frequency of establishing transgenic chickens seems to be reasonable. This frequency could be acceptable, but there are several possibilities to further improve the efficiency. One is to reduce the possible damages of donor PGCs by, for example, the use of alternative methods such as density gradient centrifugation with Ficoll (26) or Nycodenz (34). The other is extensive depletion of endogenous PGCs. For this, several strategies may be available. For instance, we will be able to remove the increasing amount of blood without severe reduction of the viability of embryos (17). Sterilization of endogenous PGCs with ultraviolet (35) or γ-ray (36) may be the alternative choice. Until now, transgenic progeny have been screened from G0 male chickens in almost all studies (7-9, 12, 13), since more chicks can be
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FIG. 6. Analysis of transgenic G1 chicken produced with lentiviral vector. (A) Representative results of mating. Progeny of #128 mated with #209 are shown. Embryo #3 was confirmed to be transgenic G1 progeny. N, wild-type genomic DNA as negative control; P, plasmid DNA as positive control. (B) Southern blotting analysis of blood (#205-1) or embryonic bodies (#205-2 and #225-1) of transgenic chickens. Two transgenic progeny (#1 and #2) were from #205 female, and one from #225 female. (C, D) eGFP expression in the blood of a G1 transgenic chicken (#205-1). (C) Blood cells of either non-transgenic (WT, left) or transgenic (right) chickens were analyzed by FACS. (D) Blood cells of transgenic chicken (#205-1) were observed using a fluorescent microscope. Left, fluorescence image; right, phase contrast. Small blood cells (indicated by arrowheads), but not erythrocytes (indicated by asterisks), expressed eGFP. Scale bars indicate 10 μm. (E) eGFP expression in a piece of the embryonic body of G1 transgenic chicken (#205-2). Left, non-transgenic chicken; right, transgenic chicken. Scale bars indicate 200 μm.
obtained by artificial insemination compared with G0 female chickens. However, our study suggests that female chickens can be a good source of G1 progeny. We observed that the percentage of donorderived PGCs tended to be higher in female recipients than in male recipients when recipient embryos were individually examined (Fig. 4). Naito et al. reported that the percentage of progeny derived from donor PGCs was dependent on gender (17, 18). We need further investigation to clarify the reason why male chimeras show lower efficiency of transmission than females.
According to Southern blotting, it appears that the integration sites of the transgene in the two progeny from #205 female were the same. Since integration of HIV and HIV-based vectors in chromosome occurs at random, except that they favor actively transcribed regions (37), it is unlikely that these two transgenic chickens are derived from two different PGCs in which independent integration events occurred in the same site. Instead, these two transgenic offspring probably derived from ova differentiated from the same PGC by cell division, suggesting that a minimal number of
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TABLE 1. List of representative G0 chickens with lentivirus-infected gPGCs. No. #128 #205 #209 #225 #226 #511 #513 a
Sex a
gPGCs injected per embryo
MOI
Number of progeny examined
G1 (%)
F F M F F F F
1600 1200
55 54
850
77
1500
24
36 30 101 30 33 34 32
0 2 (6.66) 0 1 (3.33) 0 0 0
F, female; M, male.
gPGCs may develop to matured ova. Overall, we could obtain transgenic chickens using gPGCs and a lentiviral vector with a substantially acceptable frequency. ACKNOWLEDGMENTS We thank the Radio Isotope Research Center of Nagoya University for supporting the radioisotope experiments, and Co60 radiation facility of Nagoya University for sterilization of host eggshells. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) by Bio-oriented Technology Research Advancement Institution (BRAIN). References 1. Houdebine, L. M.: Transgenic animal bioreactors, Transgenic Res., 9, 305–320 (2000). 2. Ivarie, R.: Avian transgenesis: progress towards the promise, Trends Biotechnol., 21, 14–19 (2003). 3. Raju, T. S., Briggs, J. B., Borge, S. M., and Jones, A. J.: Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branchspecific galactosylation and importance for engineering recombinant glycoprotein therapeutics, Glycobiology, 10, 477–486 (2000). 4. Salter, D. W., Smith, E. J., Hughes, S. H., Wright, S. E., and Crittenden, L. B.: Transgenic chickens: insertion of retroviral genes into the chicken germ line, Virology, 157, 236–240 (1987). 5. Bosselman, R. A., Hsu, R. Y., Boggs, T., Hu, S., Bruszewski, J., Ou, S., Kozar, L., Martin, F., Green, C., and Jacobsen, F., et al.: Germline transmission of exogenous genes in the chicken, Science, 243, 533–535 (1989). 6. Kwon, M. S., Koo, B. C., Choi, B. R., Lee, H. T., Kim, Y. H., Ryu, W. S., Shim, H., Kim, J. H., Kim, N. H., and Kim, T.: Development of transgenic chickens expressing enhanced green fluorescent protein, Biochem. Biophys. Res. Commun., 320, 442–448 (2004). 7. Kamihira, M., Ono, K., Esaka, K., Nishijima, K., Kigaku, R., Komatsu, H., Yamashita, T., Kyogoku, K., and Iijima, S.: High-level expression of single-chain Fv-Fc fusion protein in serum and egg white of genetically manipulated chickens by using a retroviral vector, J. Virol., 79, 10864–10874 (2005). 8. Harvey, A. J., Speksnijder, G., Baugh, L. R., Morris, J. A., and Ivarie, R.: Expression of exogenous protein in the egg white of transgenic chickens, Nat. Biotechnol., 20, 396–399 (2002). 9. Rapp, J. C., Harvey, A. J., Speksnijder, G. L., Hu, W., and Ivarie, R.: Biologically active human interferon α-2b produced in the egg white of transgenic hens, Transgenic Res., 12, 569–575 (2003). 10. Kodama, D., Nishimiya, D., Iwata, K., Yamaguchi, K., Yoshida, K., Kawabe, Y., Motono, M., Watanabe, H., Yamashita, T., Nishijima, K., Kamihira, M., and Iijima, S.: Production of human erythropoietin by chimeric chickens, Biochem. Biophys. Res. Commun., 367, 834–839 (2008). 11. Kyogoku, K., Yoshida, K., Watanabe, H., Yamashita, T., Kawabe, Y., Motono, M., Nishijima, K., Kamihira, M., and Iijima, S.: Production of recombinant tumor necrosis factor receptor/Fc fusion protein by genetically manipulated chickens, J. Biosci. Bioeng., 105, 454–459 (2008). 12. Lillico, S. G., Sherman, A., McGrew, M. J., Robertson, C. D., Smith, J., Haslam, C., Barnard, P., Radcliffe, P. A., Mitrophanous, K. A., Elliot, E. A., and Sang, H. M.: Oviduct-specific expression of two therapeutic proteins in transgenic hens, Proc. Natl. Acad. Sci. U. S. A., 104, 1771–1776 (2007).
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