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Published as doi: 10.1096/fj.07-101485.
 (The FASEB Journal. 2008;22:2435-2444.)
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Generation of transgenic quail through germ cell-mediated germline transmission

Sang Su Shin*,1, Tae Min Kim*,1, Sun Young Kim*, Tae Wan Kim*, Hee Won Seo*, Seul Ki Lee{dagger}, Se Chang Kwon{ddagger}, Gwan Sun Lee{ddagger}, Heebal Kim*, Jeong Mook Lim* and Jae Yong Han*,2

* Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea;

{dagger} Avicore Biotechnology Institute, Incorporated, Gyeonggi-Do, Korea; and

{ddagger} Hanmi Research Center, Hanmi Pharm Company, Limited, Gyeonggi-Do, Korea

2Correspondence: Department of Agricultural Biotechnology, Seoul National University, Seoul, 151–921, Korea. E-mail: jaehan{at}snu.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Here, we describe the production of transgenic quail via a germline transmission system using postmigratory gonadal primordial germ cells (gPGCs). gPGCs retrieved from the embryonic gonads of 5-day-old birds were transduced with a lentiviral vector and subsequently transferred into recipient embryos. Testcross and genetic analyses revealed that among three germline chimeric G0 quail, one male produced transgenic offspring; of 310 hatchlings from the transgenic germline chimera, 24 were identified as donor-derived offspring, and 6 were transgenic (6/310, 1.9%). Conventional transgenesis using stage X blastodermal embryos was also conducted, but the efficiency of transgenesis was similar between the two systems (<1.6 vs. 1.9% for the conventional and gPGC-mediated systems, respectively). However, substantial advantages can be gained from gPGC-mediated method in that it enables an induced germline modification, whereas direct retroviral transfer to stage X embryos causes mosaic integration. The use of gonadal PGCs for transgenesis may lead to the production of bioreactors.—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., Han, J. Y. Generation of transgenic quail through germ cell-mediated germline transmission.


Key Words: primordial germ cells • lentiviral vector • avian transgenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BIRDS HAVE ADVANTAGES OVER OTHER vertebrates as research models, in that experimental treatments can be applied at all stages of embryo development, and subsequent analyses are possible in ovo (1) . In addition, their reproductive attributes make them suitable candidates for efficient bioreactors; thus, many researchers have focused on creating new methods for transgenesis in birds (2) . Successful avian transgenesis has been achieved using a Moloney murine leukemia virus (MoMLV)-based retroviral vector, which was transduced into an undifferentiated stage X embryo (3 , 4) ; however, the efficacy of this method is limited by such problems as low efficiency and transgene silencing (4 , 5) . In its place, lentiviral vectors have gained recent favor for various gene transfer purposes, as they can stably integrate into the host genome via its innate nuclear import mechanism (6) ; in fact, lentiviral vectors are currently used in gene therapy and transgenesis (7 8 9 10 11 12 13) . Various classes of lentiviral vectors have recently been applied to stage X embryos to produce transgenic birds that express their transgenes in a ubiquitous (10 , 11) or tissue-specific manner (12 , 13) .

Primordial germ cells (PGCs) in birds are recognized as an efficient tool for achieving transgenesis, as they display a unique migration pathway via the circulation toward the primary sex cord (14 15 16 17) . We previously established a germline chimera production system by transferring postmigratory gonadal PGCs (gPGCs) into the blood of heterologous recipient embryos in both chicken and quail (18 , 19) . The gPGCs used for the transplantation were efficiently maintained in vitro without losing their germ-line competency, which shows the usefulness of PGC-mediated systems in inducing stable genetic modifications (20) . Moreover, the production of transgenic chickens by genetic modification of circulatory PGCs obtained from stage 14–16 embryos was recently reported (17) .

Quail are useful model organisms for research in development and genetics. They offer numerous advantages, including a short generation interval, disease resistance, and high laying capacity (21) . In this study, we derived transgenic quail using gPGCs and a lentiviral vector, and we report the first-ever production of transgenic quail germline chimeras.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal care and general experimental procedures
All procedures for animal management and reproduction were performed in accordance with the standard protocols of Seoul National University. The Institutional Review Board of the Department of Animal Science and Technology, Seoul National University, approved the research proposal and all relevant experimental procedures in January 2002. Quail were maintained at the University Animal Farm, College of Agriculture and Life Sciences, Seoul National University. Our general experimental procedures are summarized in Fig. 1 .


Figure 1
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  		Figure 1. General procedure for the production of transgenic quail. Germline modification was induced by two different methods: transplantation of ex vivo transfected primordial germ cells collected from stage 28 wild-type plumage (WP) embryos into the dorsal aorta of a stage 13 black strain Japanese quail embryo (A), or direct lentiviral gene transfer into stage X blastodermal WP embryos (B).

Vector construction and lentiviral vector production
The 1.9-kb fragment digested from plasmid pWPI with KpnI and ClaI was ligated into pLenti4/V5GW/LacZ (Invitrogen, Carlsbad, CA, USA) using the same restriction sites. The plasmid was then excised with KpnI and ligated with the 1.6-kb fragment from pWPI, which was digested with KpnI, resulting in pLTEiGW. The Rous sarcoma virus (RSV) promoter was derived from pLXRN (Clontech, Palo Alto, CA, USA) by digestion with HindIII and cloned into the HindIII site of pBluescriptIISK(–) (Stratagene, La Jolla, CA, USA), creating pBSSK-RSV. The elongating factor-1 (EF-1) alpha promoter of pLTEiGW was replaced with the RSV promoter retrieved from the ClaI/EcoRI fragment of pBSSK-RSV, resulting in the clone pLTRiGW. The final transfer vector pLTReGW was generated by deleting IRES-GFP with PmeI/NheI and inserting the eGFP fragment obtained from pEGFP-1 (Clontech) digested with SmaI and XbaI. The schematic drawing of the vector is depicted in Fig. 2 .


Figure 2
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  		Figure 2. Schematic representation of the pLTReGW vector. Restriction sites EcoRI (EI) and EcoRV (EV) and the location of the probe used for Southern blot analysis are depicted. Arrows and arrowheads indicate the primers used to amplify 1.2-kb and 410-bp fragments in the proviral construct, respectively. LTR, long terminal repeat; {Psi}, packaging signal; RRE, Rev-response element; RSV, RSV promoter; cPPT, central polypurine tract of HIV-1; EGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. Scale bar = 1 kb.

Lentiviral vector particles were produced by calcium phosphate coprecipitation method. Briefly, 5 x 106 293FT cells (Invitrogen) were plated on a 100-mm culture dish in Dulbecco’s minimal essential medium (DMEM; Life Technologies, Invitrogen, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Life Technologies, Invitrogen) on the day before transfection. On the day of transfection, 10 µg of pLTReGW and 10 µg of Virapower packaging mixture (Invitrogen) were added to a final volume of 450 µl of 0.1x TE (1 mM Tris-Cl+0.1 mM EDTA, pH 7.6). After the addition of 50 µl of 2.5 M CaCl2, 500 µl of 2x HEPES-buffered saline (281 mM NaCl, 100 mM HEPES, 1.5 mM Na2HPO4, pH 7.0) was added and gently mixed by bubbling. The DNA mixture was then incubated at room temperature for 10 min and subsequently added to the 293FT cells. After 16 h of transfection, the medium was renewed with 4 ml of DMEM + 10% FBS. The supernatant was harvested after 48 h, filtered through 0.22-µm pore-size cellulose acetate filters and then concentrated to 100x by centrifugation at 26,000 rpm for 100 min with an ultracentrifuge (XL-90, Beckman Coulter, Fullerton, CA, USA). To determine its titer, nonconcentrated viral supernatant was serially diluted and subsequently transfected to 3 x 105 quail embryonic fibroblasts in a 6-well cell culture plate (TPP, Trasadingen, Switzerland), which resulted in 1–10 x 106 TU/ml of titer by end point dilution titration (data not shown).

Lentiviral gene transfer into stage X blastodermal embryos
Freshly laid eggs from wild-type plumage (WP: d+/d+) Japanese quail were wiped with 70% ethanol and positioned with their sides facing upward for at least 4 h at room temperature. A window (5 mm in diameter) was then made using fine tweezers, and 2–3 µl of concentrated lentiviral particle soup was injected into the subgerminal cavity of the blastoderm. The window was then sealed twice with paraffin film, and the egg was incubated with the pointed end down until hatching.

Lentiviral gene transfer and transplantation of primordial germ cells into stage 13 recipient embryos
Quail with wild-type (WP: d+/d+) and black (D: homozygous for the autosomal incompletely dominant gene D) plumage were used as gPGC donors and recipient embryos, respectively (19) . To purify gPGCs, a MiniMACS system (Miltenyi Biotec, Auburn, CA, USA) was used to purify gPGCs from a mixed population of gonadal cells by magnetic activated cell sorting (MACS) before microinjection into recipient embryos (17) . In brief, freshly isolated quail gonadal cells (1x106) from stage 28 embryos were incubated with the monoclonal antibody QCR1 for 20 min at room temperature. After washing with 1 ml of Ca2+- and Mg2+-free phosphate-buffered saline [PBS(–)] containing 0.5% (w/v) bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) and 2 mM EDTA (Sigma), the cell pellet was mixed and incubated for 15 min at 4°C with 100 µl of PBS(–) containing 20 µl of microbeads coated with goat antibodies to mouse IgG. The cells were then washed with 500 µl of PBS(–) and loaded into the MiniMACS. For the purification of gPGCs by EDTA, freshly isolated quail gonads were incubated in PBS(–) containing 0.5 mM EDTA for 20 min at room temperature, followed by gentle kneading in PBS(–) containing 0.4% BSA with a fine glass needle under x200 viewing. A cell suspension containing gPGCs was harvested using a micropipette. To determine the proportion of gPGCs, 5 x 103 cells were cultured in DMEM supplemented with 10% FBS for 8 h and stained with QCR1. Next, the cells were fixed in 1% (w/v) glutaraldehyde (Sigma) for 5 min and washed twice in PBS(–), before being stained with QCR1 [1:200 dilution in PBS(–)], which is specific for quail PGCs (19) , using a Universal LSAB peroxidase kit (Dako Cytomation, Carpinteria, CA, USA). The gPGCs were then observed using an inverted microscope (Eclipse-300; Nikon, Tokyo, Japan). In each transfection, gPGCs were subjected to lentiviral transduction at a multiplicity of infection (MOI) of 30 or 100 using concentrated vector dissolved in DMEM supplemented with 10% FBS for 6 h at 37°C. For cell transplantation, a small window was made at the pointed end of the recipient egg, and ~2 µl (containing 10,000 or 5000 gonadal cells at an MOI of 30 and 100, respectively) were injected into the upper portion of dorsal aorta of the stage 13 embryo (50-h incubated) using a micropipette. The window was then sealed twice with paraffin film, and the egg was incubated with the pointed end down until hatching.

Mating and testcross analysis
All G0 quail produced by direct lentiviral gene transfer were mated to nontransgenic wild-type plumage (d+/d+) quail, except for one case in which two G0 quail were mated to each other, in order to minimize the generation time of homozygous transgenic quail. All G1 hatchlings were then subjected to transgene detection analysis.

For G0 quail produced by the PGC-mediated method, donor gamete-derived offspring with wild-type plumage (d+/d+) were identified on the basis of feather color and selected from the total offspring (D/d+ and d+/d+). The efficiency of germline transmission of donor gPGCs was determined as the percentage of offspring having wild-type plumage (d+/d+) among the total offspring (D/d+ and d+/d+). All donor gamete-derived G1 hatchlings (d+/d+) were then subjected to transgene detection analysis.

Detection of the transgene in quail by PCR and Southern blot analysis
As the population of G0 founders produced by direct lentiviral gene transfer was small, no procedure for prescreening G0 transgenic founder was conducted. To confirm the integration of the transgene in G1 and G2 quail, genomic DNA was extracted from the blood of hatched quail using a Puregene DNA purification kit (Gentra Systems, Minneapolis, MN, USA). Two primer sets for the 1.2-kbp and 410-bp fragments were used to detect the transgene by PCR. The PCR was performed in a total volume of 25 µl, containing 200 ng of genomic DNA, 10 mM dNTP (Corebio, Seoul, Korea), 2.5 µl of 10x reaction buffer (Corebio), 0.4 µM sense and antisense primers (Bioneer, Daejon, Korea), and 0.4 U of DNA polymerase (Corebio) using a Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany). For negative and positive controls, genomic DNA extracted from nontransgenic quail and 4 pg of pLTReGW plasmid DNA was used as the template, respectively. For the primer sequences for the amplification of the 1.2-kbp and 410-bp fragments and PCR parameters, see Supplemental Table 1 . For Southern blot analysis, 10 µg of genomic DNA from G1 quail was digested with EcoRI or EcoRV. The digested DNA was electrophoresed on 1.0% agarose gel and then transferred to a nylon membrane (Hybond-N+; Amersham Biosciences, Piscataway, NJ, USA). The blot was hybridized with a 32P-labeled eGFP probe prepared by a Random Primer DNA Labeling Kit (Takara Bio Inc., Shiga, Japan). Hybridization signals were detected by autoradiography.


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  		Table 1. Generation of transgenic quail by transplantation of ex vivo transfected gPGCs into recipient embryos or transfusion of a lentiviral vector into stage X blastodermal embryos

Identification of transgene flanking regions
The sequences flanking the transgene integration site were identified using DNA Walking SpeedUpTM Premix Kit-II (Seegene, Seoul, Korea) according to the user’s manual. To clone the 5' flanking region, three oligomers for the target specific primer (TSP) were designed. The sequences of TSP 1, TSP 2, and TSP 3 are listed in Supplemental Table 1 . The first PCR was performed in a total volume of 25 µl, containing 100 ng of genomic DNA extracted from the blood of transgenic quail, 12.5 µl of 2x reaction buffer, 0.4 µM TSP1, and 0.2 µM each of the DW-ACP-1, 2, 3, and 4 primers. A 3-µl aliquot of the PCR product was then used as a DNA template for the second PCR. The PCR reaction was performed in a total volume of 20 µl, containing 0.5 µM TSP2 primer, 0.5 µM ACPN, and 10 µl of 2x reaction buffer. After confirming amplification by gel electrophoresis, the final round of PCR was conducted by amplifying 1 µl of amplified product from the second PCR in a total volume of 20 µl, consisting of 0.125 µM universal primer, 0.5 µM TSP3 primer, and 2x reaction buffer. The PCR parameters were as follows. 1) First PCR: 94°C for 5 min, 42°C for 1 min, 72°C for 2 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 100 s; final extension at 72°C for 5 min. 2) Second PCR: 94°C for 5 min; 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 60 s; final extension at 72°C for 7 min. 3) Third PCR: 94°C for 3 min; 30 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 60 s; final extension at 72°C for 5 min. After the third PCR, a fragment 200–300 bp shorter than the second PCR fragment was extracted from 1.0% agarose gel, purified using a PCR purification kit (Geneall, Seoul, Korea), cloned into a pGEM-T easy vector (Promega, Madison, WI, USA), and sequenced using an ABI Prism 3730 XL DNA Analyzer (PE Applied Biosystems, Foster City, CA, USA). The sequences of the 5' flanking region were analyzed using the UCSC genome browser (http://genome.ucsc.edu). Once the sequence of the 5' flanking region was identified, an additional oligomer priming the upstream of the integration site was designed for further sequence confirmation (see Supplemental Table 1 for primer sequences). PCR was performed in a total volume of 25 µl, containing 200 ng of genomic DNA isolated from G1 transgenic quail, 10 mM dNTPs, 2.5 µl of 10x reaction buffer, 0.4 µM newly designed primer, 0.2 µM TSP3, and 0.4 U of DNA polymerase. The thermal cycling profile for PCR was as follows: 94°C for 5 min (initial denaturation); 35 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min; final extension at 72°C for 5 min. The amplified products were electrophoresed in 1.0% (w/v) agarose gel, purified, and sequenced.

Fluorescence in situ hybridization (FISH)
Quail embryonic fibroblasts (QEFs) were prepared from stage 28 transgenic embryos produced from G1 transgenic quail or between two G2 hemizygous transgenic quail. After cells reached 70% confluency, they were incubated with 0.05% colchicine (Sigma) for 50 min, treated with 0.8% sodium citrate, and dropped onto a glass slide. The slides were then fixed (methanol:acetic acid=3:1) and dried for 1 wk. A probe was prepared using a Biotin Nick Translation Mix kit (Roche, Basel, Switzerland) according to the user’s guide. After the labeled probe was denatured in a mixture with salmon sperm DNA, Human Cot-1, and Hyb for 10 min at 75°C, the mixture was hybridized overnight with chromosome specimens that had been denatured in 70% formamide/2x saline-sodium citrate (SSC) at 75°C for 5 min. The slides were then washed, blocked with 4% BSA, and incubated with 5 µg/ml fluorescein avidin D, cell sorter grade (DCS; Vector Laboratories, Burlingame, CA, USA) at 37°C for 20 min. The slides were then washed with 4x SSC/0.1% Tween 20 at 45°C for 5 min, and incubated with 5 µg/ml ImmunoPure biotinylated goat anti-avidin (Pierce, Rockford, IL, USA) at 37°C for 20 min. After washing, the specimens were incubated with fluorescein avidin DCS at 37°C for 20 min. Finally, the slides were washed, dehydrated, and counterstained with 100 ng/ml 4',6-diamidino-2-phenylindole (DAPI; Sigma). Microscopic images were captured using a Leica DMRXA2 (Leica Microsystems GmbH, Wetzlar, Germany).

Expression analysis
A total of three 3-wk-old heterozygous transgenic G2 quail, two from direct lentiviral infection and one from gPGC-mediated method, respectively, were euthanized by CO2 inhalation, and their organs were fixed overnight in 4% paraformaldehyde (Sigma). After washing in PBS(–) several times, they were infused with 30% sucrose (Sigma) for 6 h, imbedded in OCT compound (Sakura Finetek U.S.A., Torrance, CA, USA), and sectioned at 14 µm using a cryostat (HM 505E; Microm, Walldorf, Germany). After the cell nuclei were counterstained with 100 ng/ml DAPI (Sigma), each section was visualized under a fluorescence microscope, and the images were recorded with an AxioVision imaging system (Zeiss, Jena, Germany). For in vivo and organ imaging of eGFP, its expression in transgenic quail was examined using the GFsP-S lens system (BLS Ltd, Budapest, Hungary).

Statistical analysis
All numerical data were analyzed with analysis of variance (ANOVA) using the general linear model (PROC-GLM) of the SAS program (SAS Institute, Cary, NC, USA). Differences were considered significant at a value of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of transgenic germline chimeras and germline transmission of a transgene to G1 quail
A similar number of gPGCs was obtained by magnetic activated cell sorting (MACS) and EDTA, as determined by staining with the quail PGC-specific antibody QCR1 (Supplemental Table 2 ). As shown in Table 1 , of 187 embryos injected with gPGCs transfected at an MOI of 30, 78 hatched (41.7%), whereas 9 hatched from 133 embryos injected with gPGCs transfected at an MOI of 100 (6.8%). The hatchlings were reared for up to 2 months, and a similar proportion of sexually mature founder quail was obtained at the two MOI levels [46/78 (59.0%) and 4/9 (44.4%) for an MOI of 30 and 100, respectively]. Testcross analysis revealed that 18 of 46 (39.1%) and 3 of 4 (75.0%) G0 founders were germline chimeras at an MOI of 30 and 100, respectively. Genetic analyses were subsequently conducted using the donor-derived offspring. Among three germline chimeras from an MOI of 100, one male produced 6 transgenic offspring out of 24 donor-derived offspring (25%), whereas no transgenic quail were produced from the germline chimeras from an MOI of 30 (Tables 1 and 2 ).


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  		Table 2. Efficiency of germline transmission and transgenesis in G0 putative germline chimeras produced by transfusion of gPGCs transfected with lentiviral vector

Of 706 stage X embryos injected with vector particles, 101 hatched (14.3%), and among these, 64 survived to adulthood and were mated with other mosaic G0 quail or with nontransgenic quail (Tables 1 and 3 ). Consequently, 7 transgenic G1 transgenic quail were generated from 5 G0 founders. The ratio of germline transmission was in the range of 0.6–1.6% (Table 3) .


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  		Table 3. Production of G1 transgenic quail from G0 founders produced by direct lentiviral gene transfer into stage X embryos

Identification of copy number and the integration sites of the transgene
Southern blot analysis of genomic DNA from the G1 transgenic quail revealed that all had a single copy of the transgene (Fig. 3 E, F). This result was also shown by the Mendelian germline transmission to G2 offspring (40.9 to 48.6%, Table 4 ). Six G1 quail from a G0 germline chimeric founder (ID name: K1) produced by PGC transplantation (ID names: TQ 8, 9, 10, 11, 12, and 13) had the same restriction fragments (Fig. 3E ), whereas different fragments were observed for each of the G1 quail (TQ 1, 2, 3, 4, and 6) produced by direct lentiviral gene transfer (Fig. 3F ).


Figure 3
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  		Figure 3. Verification of transgenesis in transgenic quail by PCR and Southern blot analysis. A) PCR analysis of G1 offspring from the G0 founder produced by direct gene transfer into stage X embryos. Two primer sets were used to produce a 1.2-kb fragment (bottom) and a 410-bp fragment (top). DNA ladders of 100 bp and 1 kb (MW) are shown in the upper and lower panels, respectively. Lanes 1–7 represent the transgenic quail TQ 1–7. Lanes 8 and 10 represent blank (distilled water) and genomic DNA from a nontransgenic quail, respectively, while 20 pg of pLTReGW was used as a positive control (Lane 9). B) Detection of transgenic G1 quail among donor-derived offspring produced from ex vivo transfected gPGCs. PCR was conducted using genomic DNA extracted from 24 wild-type plumage (d+/d+) G1 quail selected by feather color out of 310 offspring produced by cross-breeding one male G0 germline chimera (K1, with D+/d+ germ cells) with a wild-type plumage female quail. Lanes 1–24 are G1 offspring that originated from donor gPGCs, while lanes 4, 12, 14, 20, 23, and 24 are transgenic quail. DNA ladders of 100 bp and 1 kb (MW) are shown in the upper and lower panels, respectively. Lane 25, blank (distilled water); Lane 28, 20 pg of pLTReGW. Lanes 26 and 27, nontransgenic male and female wild-type plumage quail, respectively. C–F) Southern blot analysis of genomic DNA extracted from transgenic G1 and G2 quail. C) Genomic DNA (10 µg) was digested with EcoRI and hybridized with the eGFP probe. Lane 1, lambda DNA/HindIII marker (20 ng); lanes 2 and 3, positive controls (500 and 50 pg of pLTReGW, respectively); lane 4, nontransgenic quail; lanes 5–10, G1 transgenic quail TQ 8, 9, 10, 11, 12, and 13, respectively; lane 11, G2 transgenic quail from G1 quail TQ 8. D) Genomic DNA (10 µg) was digested with EcoRI. Lanes 1–5, G1 transgenic quail TQ 1, 2, 3, 4, and 6, respectively; lane 6, nontransgenic quail. E) Genomic DNA (10 µg) was digested with EcoRV. Lane 1, lambda DNA/HindIII marker (20 ng); lane 2, nontransgenic quail; lanes 3–8, G1 transgenic quail TQ 8, 9, 10, 11, 12, and 13, respectively; lane 9, one G2 transgenic quail from G1 quail number TQ 8; lane 10, positive control (500 pg of pLTReGW). F) Genomic DNA (10 µg) was digested with EcoRV. Lane 1, lambda DNA/HindIII marker (20 ng); lane 2, nontransgenic quail; lanes 3–7, G1 transgenic quail TQ 1, 2, 3, 4, and 6, respectively.


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  		Table 4. Production of G2 transgenic offspring from G1 transgenic quail

Sequencing and FISH analysis revealed that the location of the transgene in six pedigrees from G0 germline chimeric founder K1 produced by the PGC-mediated method was in the same region of chromosome 5 (Fig. 4 A, C; Supplemental Table 3 ). This indicates that these G1 quail originated either from the same transgenic PGC or from different gPGCs in which the transgene had inserted into the same chromosomal location. Of the seven pedigrees generated by direct lentiviral infection, three G1 quail (TQ 1, 5, and 7) produced from the same G0 founder had the same integration locus as those produced by PGC-mediated transgenesis, whereas the other four (from different G0 founders) had different integration sites (chromosomes 3, Z, 4, and 3 in TQ 2, 3, 4, and 6, respectively; Fig. 4 and Supplemental Table 3 ). The probability of the observed number of site-specific integrations (x) from n independent trials was P = 1.04e-17 using the exact binomial probability. We presupposed that the six G1 quail produced by PGC-mediated transgenesis and the three G1 quail generated by direct lentiviral gene transfer into stage X blastodermal embryos originated from a single integration event in each case, because it is more likely than assuming that they originated from different integration events. To investigate the pattern of each integration event, a 648-bp region spanning quail genomic DNA upstream of the integration site was sequenced. As a result, three high-quality single nucleotide polymorphisms (SNPs) were found using the Phred/Phrap/Consed package (University of Washington, Seattle, WA, USA; www.phred.org). On the basis of our SNP data, we confirmed that TQ 11 and TQ 8 had unique alleles 376 and 198 bp upstream of the integration site, respectively, among the six G1 transgenic quail produced by PGC-mediated transgenesis. Among the three G1 quail produced by direct lentiviral gene transfer (TQ 1, 5, and 7), a unique allele was detected in TQ 7 at 240 bp upstream of the integration site. The probability of site-specific integrations (x=5) from the independent trials (n=9) was recalculated to P = 5.06e-44; thus, it is highly unlikely that these integrations occurred by chance.


Figure 4
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  		Figure 4. Transgene mapping by DNA sequencing and FISH. A) Amplification of the upstream transgene-flanking region in transgenic quail (TQ 8–13). All quail had the same 648-bp fragment. Lanes 1–6 represent TQ 8–13; lanes 7 and 8 represent a nontransgenic quail and a blank (distilled water), respectively. B) Amplification of the upstream transgene-flanking region of transgenic quail. Lanes 1–7 designate TQ 1–7. C–F) Transgene mapping by FISH. The transgene was mapped to chromosome 5 in TQ 8 (C) and TQ 1 (D), chromosome 3 in TQ 2 (E), and chromosome Z in TQ 3 (F). All samples were prepared from stage 28 embryo fibroblasts of G2 hemizygous transgenics, except one chromosome spread from a G3 homozygous transgenic from TQ 1 (D). A fluorescein-labeled probe and DAPI were used for transgene detection and chromosome counterstaining, respectively. Arrowheads indicate the location of the transgene in the chromosome. Image view x1200 (C–F).

Analysis of transgene expression
By both methods, a similar expression pattern was noted in various tissues. Strong eGFP expression was detected in the wing muscle, thigh muscle, pectoral muscle, liver, and villi of the small intestine (Fig. 5 ). The level of expression differed by organ and even within the same tissue (Fig. 5K-T ).


Figure 5
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  		Figure 5. Confirmation of transgenesis in transgenic quail based on enhanced green fluorescent protein (eGFP) expression. Expression analysis was conducted using three 3-wk-old G2 heterozygous transgenic quail, two from quail produced by direct gene transfer into stage X blastodermal embryos (C, F, I, N, O, S, T for TQ 2–21 and E, G, J, K, L, M for TQ 1–45, respectively) and one from gonadal primordial germ cell-mediated method (D, H, P, Q, and R for TQ 8–33). A) Germline chimeric G0 male Japanese quail. B) Nontransgenic wild-type plumage female quail employed for testcross analysis. C–H) Fluorescence illumination of transgenic quail. eGFP expression was detected in the head (C, D), wing (E), pectoral muscles (F, G) and thigh muscles (H). I–M) Detection of eGFP expression in the skeletal muscles and internal organs. eGFP expression was detected in the pectoral muscle (J), gastrointestinal tract (K), heart (L), and liver (M). N–T) Direct fluorescence images of frozen sections of organs from transgenic quail. eGFP expression was detected in the thigh muscle (N, O), pectoral muscle (P), large intestine gland (Q), the villi of the small intestine (R, S) and the liver (T).

Evaluation of gPGC-mediated transgenesis
A comparison of our results obtained by PGC-mediated and conventional transgenesis is shown in Table 5 . The transgenesis efficiency was similar, however, gPGC-mediated transgenesis offers substantive advantage for designed gene manipulation, because gPGCs can be genetically modified and maintained in vitro for induced germline modification.


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  		Table 5. Comparison between direct lentiviral gene transfer into stage X blastodermal embryos and gPGC-mediated method for transgenesis in quail


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we generated transgenic Japanese quail via the transplantation of lentivirally transduced gPGCs from quail with wild-type plumage (WP: d+/d+) into black plumage (D: homozygous for the autosomal incompletely dominant gene D) quail embryos. This achievement confirms the availability of a PGC-mediated, germline chimera production system for generating transgenic birds (18 19 20) .

Until recently, retroviral gene transduction into stage X blastodermal embryos was a steadfast method for modification of the germline, because it involves early undifferentiated embryonic cells, including PGCs (4 , 5) . To evaluate the availability of a PGC-mediated method, we attempted this conventional method in parallel. Various parameters were subsequently monitored, and a comparison was made between PGC-mediated transgenesis and direct lentiviral gene transfer into stage X blastodermal embryos. The efficiency of transgenesis did not differ between the two methods; however, substantial advantages are offered by the PGC-mediated method in that it has unlimited potential for establishing an efficient system for targeted genetic manipulation, rather than provoking mosaic integration into the early undifferentiated cells of stage X embryos. Furthermore, putative transgenic G1 offspring can be easily preselected by observing its feather color.

Our data suggest that an HIV-1-based lentiviral vector might occupy specific loci in order to maintain its proviral integrity in quail gPGCs, although it has been shown to integrate into the thousands of active transcriptional units spread across the chromosomes in chicken somatic cells, particularly those within microchromosomes (23) . However, our results oppose this trend. The transgene distribution observed here was restricted to macrochromosomes (autosomes 2–5 and sex chromosome Z), in which the gene density was low compared to that in the microchromosomes (24) . Therefore, it is conceivable that during gametogenesis, ablation might have occurred in those cell lineages that originated from microchromosomally inserted gPGCs because of the inactivation of nearby active genes (25) . Although not sufficient, our results provide an important resource for developing a smart vector for PGC-based germline modification in avian species by maximizing the number of viable gPGCs carrying the transgene. Our results regarding the integration site of the lentiviral vector should facilitate the development of an effective avian transgenic system based on a nonviral vector, which is important for the production of bioactive materials using a bioreactor system.

Another important issue can now be addressed in relation to the location of the transgene. In one report on chicken (26) , a transgene was located in gene-dense (micro) chromosome 26, and a somewhat higher and consistent expression pattern was detected compared to our results, even though the same gene cassette (RSV promoter-eGFP) was used. Within this context, the level of transgene expression may be correlated with the characteristics of the regions flanking the transgene (e.g., gene density; 24, 27). In addition to the integration site of the vector, the transgene copy number significantly affects its expression level. Such a correlation was previously shown in mice; transgene expression in animals carrying two or more copies was high but was undetectable in animals carrying a single copy (8) . Similarly, other studies have suggested that consistent transgene expression requires more than two copies of the lentiviral vector construct (9 , 28) . Apart from these effects, other factors such as the genetic background of the animals or inbreeding depression might have caused inconsistent expression of the transgene (29) . To fully elucidate the regulatory mode of transgene expression, other relevant approaches such as epigenetic analysis should be applied.

A comparison of the biological aspects before and after germline transmission of the conventional and PGC-mediated methods will facilitate the development of a transgenic system capable of using cells at various developmental stages; consequently, sequential gene targeting by inserting or deleting genes at a specific locus in developing cells may be possible at any point during embryogenesis and differentiation. Thus, the results of this study may lead to the development of transgenic bioreactors or a humanized avian model.

In summary, our study demonstrates that ex vivo manipulated gPGCs can differentiate into transgenic gametes in the gonads of a recipient embryo and that the resulting chimera can successfully produce transgenic offspring. Combined with genetic and cellular manipulations, this unique transplantation model using postmigratory gPGCs will benefit various research sectors in medicine and biotechnology. Our PGC-mediated method avoids mosaic integration by using stable cell lines and is capable of preselecting donor-derived offspring by simple parameters. However, no evidence has been reported that describes a reliable route for inducing intended changes in the quail genome. Also, the lack of information regarding whether gPGCs can accept DNA while retaining the attributes of germ cells is problematic because the in vitro manipulation of gPGCs may negatively impact their function. Nevertheless, the creation of state-of-the-art technologies for the site-specific alteration of avian gPGCs will aid the production of humanized bioreactors or model birds by allowing for selective germline modification. Analysis of the new transgenic pedigrees that will soon be established will promote the establishment of a novel germ cell-based method of transgenesis.


   ACKNOWLEDGMENTS
 
This study was supported by a graduate fellowship from the Brain Korea 21 project of the Korean Ministry of Education and the Research Institute for Agriculture and Life Sciences, Seoul National University. We thank D. Trono (École Polytechnique, Lausanne, Switzerland) for providing the pWPI vector and H. Aoyama (Hiroshima University, Hiroshima, Japan) for providing QCR1. Fluorescence imaging and ultracentrifugation were conducted at the National Instrumental Center for Environmental Management, Seoul National University.


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication November 6, 2007. Accepted for publication January 10, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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