Progress Toward the Culture and Transformation of Chicken Blastodermal Cells - 《stemcells》干细胞学杂志干细胞之家



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作者：Youliang Wang, Carrie F. Brooks, Steven A. Jones, Lyn K. Olliff, Michael Morgan, Gordon L. Speksnijder, Chuck Foley, Alex J. Harvey作者单位：AviGenics, Inc., Georgia BioBusiness Center, Athens, Georgia, USA
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      【摘要】2 D5 c. ~- K8 _1 R1 t% Z$ Q
   Chicken blastodermal cells can be cultured for short periods of time and retain the ability to contribute to somatic and germline tissues when injected into -irradiated stage X embryos. Such a method has yet to yield a germline transgenic bird, in part due to the low rate of transgene integration into the avian genome. In addition, the short culture period precludes the identification and expansion of those cells that carry an integrated transgene. In this study, two methods were developed that produced blastodermal cells isolated from stage X Barred Plymouth Rock embryos bearing an integrated transgene. Addition of chick embryo extract to the culture medium enabled expansion of single colonies for multiple passages. Southern blot analysis indicated that the transgenes had integrated as a single copy in most of the clones. Cells from passaged, transgenic embryo cells were injected into irradiated stage X White Leghorn embryos, producing hatched chicks that bore the donor cells in their somatic tissues. Transgene sequences were detected in sperm DNA; however, breeding of chimeras did not result in germline transmission of the transgene, indicating that the contribution of the transgenic cells to the germline was either nonexistent or very low.
      【关键词】 Avian transgenesis Blastodermal cell culture Embryonic stem cells
   INTRODUCTION

Blastodermal cells (BDCs) isolated from stage X embryos have been an attractive vehicle for avian transgenesis, yet there remain major hurdles that prevent the production of transgenic flocks of chickens. BDCs are readily isolated from freshly oviposited eggs, making them amenable to collection and direct injection into recipient embryos. Each embryo can yield tens of thousands of cells, which can be transfected by numerous techniques. Most importantly, the cells can contribute to somatic and germline tissues upon reintroduction into recipient embryos . Even with all of these advantages, however, the introduction of DNA into manipulated BDCs has yet to produce a transgenic chicken that is able to pass the transgene through the germline. This stems from two major hurdles: the low rate at which exogenous DNAs integrate into the genome and the propensity of BDCs cultured for any significant period of time to lose the ability to contribute to germline tissues.

Injection of Barred Plymouth Rock (BPR) BDCs into a White Leghorn (WL) embryo results in chicks that bear patches of black feathers derived from the BPR cells that incorporated into the WL embryo. Donor BPR cells can also contribute to germ tissues, which can be determined by breeding of the chimeric roosters to BPR hens ." h* L1 K: P- X: X; ?$ q
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BDCs are readily transfected, typically by addition of cationic liposome/DNA complexes to the cells in culture . However, there was no evidence of persistence of the transgene in hatched chicks. Magnetically sorted cells were also able to contribute the somatic tissues, based on expression of the lacZ gene in embryos, although no chicks were hatched in this study, so it was difficult to assess whether the transgene had stably integrated.
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It is likely that a prohibitively low percentage of transfected BDCs injected into embryos bear an integrated transgene. To identify and enrich for those cells with an integrated gene, the cells may have to be cultured for a period of time that will allow segregation of stably integrated and episomal DNAs. However, embryonic cells from all species rapidly undergo differentiation during culture, thus losing the ability to contribute to germline tissues. The exception is mouse embryonic cells, which, when derived from a few select lines of mice and handled carefully, can be cultured extensively and remain able to differentiate into germ cells . The PGCs were not transfected, and thus the utility of this method for transgenesis was not assessed.7 [/ f' `7 q- ~; a

In the current study, we attempted to do several things: 1) develop methods to efficiently transform stage X BDCs via transfection of linearized plasmid DNA; 2) assess the transformation efficiency using such methods; and 3) develop culture conditions that would allow the propagation of such cells while maintaining pluripotency. Using unique combinations of transformation procedures and culture methods, we were able to transform BDCs at a rate of 1 in 100,000 harvested cells. Using cell culture media supplemented with chick embryo extract, clones of transformed blastodermal cells were expanded for multiple passages. Cells from various passages were able to contribute to somatic and possibly germ tissues when injected into recipient embryos.
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MATERIALS AND METHODS
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Animal Care  u. L( H& Q; \# p) A( ^9 c3 w. n

Birds used in this study were cared for according to the guidelines and protocols reviewed and approved by the University of Georgia Animal Care and Use Committee.$ E: R5 ^0 x7 j- V2 \' k' d9 B: A8 u
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Preparation of Feeder Cells; r" V; F- O- T4 [+ ]5 z! q3 ~

Six-well tissue cultures plates (Falcon; BD Biosciences Discovery Labware, Bedford, MA, http://www.bdbiosciences.com) were treated with 0.1% gelatin for several minutes and air-dried. STO cells (American Type Culture Collection, Manassas, VA, http://www.atcc.org) were grown to confluency in 100 mm plates in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) with high glucose, L-glutamine, sodium pyruvate, pyridoxine hydrochloride (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 10% fetal bovine serum, 0.1 mM minimal essential medium (MEM) nonessential amino acids (Invitrogen), 50 U/ml penicillin-streptomycin, 2 mM L-glutamine (Invitrogen) at 37¡ãC, 5% CO2. STO cells were treated with 10 µg/ml mitomycin C (Mitamycin; Bristol-Myers Squibb, Princeton, NJ) in STO medium at 37¡ãC, 5% CO2 for 2.5 hours. Cells were trypsinized and centrifuged, and the pellets were resuspended in STO culture media. Cells from one 10-cm plate were distributed to six 35-mm wells that had been pretreated with gelatin (approximately 1.0 x 106 cells per well). STO feeder plates were used within 1¨C2 days.1 E" [; b4 d0 J7 b7 I" S$ H! ~- f

Preparation of Linearized DNAs" _8 E8 \0 a8 e
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Supercoiled plasmid purified by cesium chloride centrifugation was linearized with restriction enzymes that cut once in the plasmid. The digests were treated with proteinase K; extracted with phenol, chloroform, and isoamyl alcohol; and precipitated with ethanol. The precipitate was spooled with a pipette tip and transferred to a new tube with 1 ml of 70% ethanol. The tube was centrifuged for 30 seconds at 3,800g, the supernatant was removed, and the pellet was air-dried. The pellet was resuspended in 4 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA overnight, and DNA was quantitated by measuring absorbance at 260 nm.# R& z% F' h. Y" z( U
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Preparation of Chick Embryo Extract
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Embryos were collected from fertilized Barred Plymouth Rock eggs incubated for 7 days. Embryos (10) were collected into a 50-ml conical tube with 10 ml of phosphate-buffered saline (PBS) (Ca2 - and Mg2 -free) and 0.15 M NaCl on ice. Cells were homogenized in a prechilled Waring blender until the 70% of the embryo was broken into single cells. The homogenate was transferred to a 50-ml conical tube, frozen in liquid nitrogen, and thawed in a 37¡ãC water bath twice. Debris was removed by centrifugation at 20,000g for 30 minutes at 4¡ãC, and the supernatant was filtered through a 0.45-µm HT Tuffryn membrane filter (Pall, East Hills, NY); samples were stored at ¨C70¡ãC until use.7 {* R8 G" t, j3 `% o

Electroporation of Blastodermal Cells" z' e1 J6 I$ N& E

BDCs were harvested from the area pellucida of several hundred Barred Plymouth Rock stage X eggs. Ten to 15 embryos were processed per 15-ml polypropylene conical tube and trypsinized essentially as described ) by gentle trituration with a 5-ml pipette. The cells were collected by low-speed centrifugation, resuspended in 2 ml of ice-cold BDC medium by gentle trituration, and counted in a hemacytometer. Cells were collected by low-speed centrifugation and resuspended to 3 x 106 cells per ml in PBS (Ca2 - and Mg2 -free). Linearized plasmid was added at 50¨C100 µg/ml, the cell/DNA mixture was incubated on ice for 10 minutes, and 800 µl was added to a 0.4-cm Gene Pulser cuvette (Bio-Rad, Hercules, CA, http://www.bio-rad.com) on ice. The cells were electroporated at 240 V and 250 µF with a Bio-Rad Gene Pulser II, and the cuvettes were incubated on ice for 20 minutes; 1 ml of ice-cold BDC medium was added to each cuvette, and the cells were transferred to a 50-ml conical tube. The cell concentration was adjusted to 1.0 x 105 cells per ml with BDC medium plus 2.5% chick embryo extract (CEE) (BDC-CEE medium), and 2.4 ml was placed in each well with STO feeder cells. The cells were cultured at 39.5¡ãC and 5% CO2 with daily changing of the medium.6 K3 z# N8 r5 {4 l. x3 o
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Isolation of Puromycin-Resistant BDC Colonies
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BDCs electroporated with a puromycin selection plasmid were cultured in BDC-CEE medium for 24 hours postelectroporation before changing with medium containing 0.5 µg/ml puromycin; medium was changed daily for 6¨C7 days. Needles for isolating colonies were formed by pulling 50-µl borosilicate micropipettes (Fisherbrand, Pittsburgh, PA) on a needle puller and breaking the tip so that the opening was approximately 300 µm. The needle was connected to a rubber tube and a CellTram (Eppendorf AG, Hamburg, Germany). The border of the colony was excised from the feeder cell layer with the sharp edge of the needle, and the colony was vacuumed into the needle and transferred to a tube. Single or mixed colonies were dissociated into single cells by repeated trituration with a P200 micropipettor (Rainin, Woburn, MA). Dissociated mixed colonies were plated onto mitomycin-treated STO cells in 24-well plates and cultured for 4¨C6 days with BDC-CEE-puromycin medium changed daily. The passage 1 cells were harvested by repeated trituration of the entire well contents with a p1000 micropipettor (Rainin), and the dissociated cells were plated onto mitomycin-treated STO cells in a 24-well plate. After 6 days, the cells were removed by trypsinization and plated on mitomycin-treated STO cells in six-well plates. Subsequent passages were into progressively larger plates. Cultures started from a single unique colony were initially plated on 96-well plates with mitomycin-treated STO cells. After 4¨C6 days, the cells were dissociated and transferred to 1¨C3 new wells of a 96-well plate with mitomycin-treated STO feeder cells. After 4¨C6 days, the cells were dissociated with a micropipettor and transferred to a well of a 24-well plate. Subsequent passages were with trypsin and into progressively larger plates.6 o  `6 |1 N% t7 |; Y$ s

Isolation of Enhanced Green Fluorescent Protein-Positive BDC Colonies( P, H+ H7 j3 P! n. m
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BDCs were electroporated with a linearized plasmid bearing the enhanced green fluorescent protein (EGFP) gene under control of the Rous sarcoma virus (RSV) promoter. The cells were plated on STO feeder cells as described in the absence of puromycin. After 4 days, homogenous green fluorescent chicken blastodermal cell colonies were identified on an Olympus IX70 epifluorescent microscope and isolated as described above. To remove contaminating, nontransgenic BDCs, homogenous EGFP-positive colonies were isolated a second time at passage 1 and transferred to new wells.

Southern Analysis of BDC Cell Lines
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BDCs derived from single puromycin-resistant or EGFP-positive colonies were cultured until 8 to 10 10-cm plates of confluent BDC colonies were available for DNA extraction, which, depending on the clone, was at passages 6 to 10. The cells were lysed in place with 100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 0.2% SDS, 200 mM NaCl, and 100 µg/ml proteinase K. DNA was extracted with phenol, chloroform, and isoamyl alcohol and precipitated with isopropanol. DNA was concentrated by centrifugation, washed with 70% ethanol, air-dried, and resuspended in water. DNA (5¨C10 µg) was digested overnight with restriction enzymes in 400 µl, concentrated by ethanol precipitation, and run on a 0.8% agarose gel. The gel was transferred to a Genescreen Plus membrane (PerkinElmer Life Sciences, Boston, http://www.perkinelmer.com) by standard methods and probed with gel-purified DNAs labeled with the Multiprime Kit (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, http://www.amershamhealth-us.com).
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Production of Chimeras+ _# M: {9 _2 z1 ?+ Y2 C; J

Freshly oviposited White Leghorn eggs were -irradiated (600 rads) and set on their side for 3¨C16 hours at room temperature. BDC colonies, along with the STO feeder layer, were treated with trypsin for 5 minutes in a 37¡ãC, 5% CO2 incubator. An equal volume of BDC medium supplemented with an additional 12.5% fetal bovine serum was added, and the colonies were dissociated into single cells by trituration. The cells were collected by low-speed centrifugation and resuspended in ice-cold BDC medium. Cells (3,000¨C12,000) were injected in a total volume of 3¨C7 µl into the subgerminal cavity of windowed eggs as described .
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Transgene Quantitation in Chimera Blood and Sperm DNA
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To facilitate detection of the transgenes in chimeric birds, a 62-base pair (bp) region of the neomycin resistance gene was inserted 3' to the 3'-untranslated region of the puromycin and EGFP expression cassettes. The 62-bp segment overlapped with the neomycin primer/probe set described in ref. 19, enabling sensitive detection of the transgenes used in this study. Blood and sperm DNA was extracted as described .! J" i! G8 X2 g9 V6 W  K' U2 o0 x
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For detection of the neor gene, primers used in the Taqman reaction were Neofor-1 (5'-TGGATTGCACGCAGGTTCT-3') and Neorev-1 (5'-GTGCCCAGTCATAGCCGAAT-3'). The Taqman probe sequence (Neoprobe) was 5'-CCTCTCCACCCAAGCGGCCG-3' and was labeled with FAM (6-carboxyfluorescin) at the 5' end and TAMRA (N,N,N',N'-tetramethyl-6-carboxyrhodamine) at the 3' end. Primers were synthesized by Invitrogen and probes by PE Applied Biosystems. Reaction conditions were as described .

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Transformation of Early Chicken Fibroblast Cells by Electroporation of Linearized Puromycin-Resistant Vectors

Initially, we developed procedures to transform primary fibroblasts derived from early chicken embryos. In our initial attempts, we attempted to electroporate fibroblasts with linearized expression vectors for the blastocidin or neomycin resistance genes. In the presence of either drug, the fibroblasts died very slowly, and drug resistant colonies rarely materialized (data not shown). When fibroblasts were treated with puromycin, the cells died within 2¨C3 days. Electroporation of a linearized vector bearing the puromycin resistance (purr) gene (pac) resulted in the appearance of a substantial number of colonies within 7¨C10 days. The percentage of cells initially electroporated that resulted in purr colonies varied from 0.1%¨C0.01%. Replacement of the cytomegalovirus promoter with the RSV promoter in the pac vector resulted in a twofold to threefold increase in the number of resistant colonies (data not shown).

Transformation of Blastodermal Cells by Electroporation of Linearized Puromycin-Resistant Vectors- d1 c; K7 y0 N. A

Based on our results with primary fibroblasts, we used similar conditions to transform BDCs. BDCs were harvested from BPR stage X embryos and immediately electroporated with linearized selection vector that contained the RSV-pac cassette at 240 V and 250 µF. The electroporation conditions were optimized by assessing transient expression of a marker gene and survival (data not shown). Cells were plated on mitotically inactivated STO cells (mouse embryo fibroblast cells) and cultured until purr colonies were evident. The concentration of puromycin was optimized to kill BDCs rapidly but not affect the STO feeder cells. Typically, for every 106 harvested BDCs, 10 colonies could be observed by 4¨C8 days of culture. BDC colonies of varying morphologies could be identified against the background of feeder cells. Most colonies had a round shape with a distinct border, referred to as type 1 colonies (Fig. 1A), or had an irregular border, referred to as type 2 (Fig. 1B). Cells tended to be small and tightly packed in both colony types. Other colonies formed into recognizable patterns, with morphologies indicative of differentiation (Fig. 1C, 1D).
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Figure 1. Morphology of puromycin resistance (purr) blastodermal cell (BDC) colonies. (A): Typical type 1 colony. (B): Typical type 2 colony. (C, D): Other colony morphologies that were consistently observed. (E): BDC colonies at passage 2 from a single purr type 2 colony. (F): BDC colonies at passage 4 from a single purr type 2 colony. Scale bar = 100 µm.: T" T! x( E1 g  k2 x+ ~6 n
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Passaging of BDC Colonies with Chicken Embryo Extract' f( g1 p- _1 b
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In initial attempts to isolate and passage BDC colonies using standard culture medium formulations and feeder cells, the majority of passaged cells did not survive, and those that did divided slowly. A number of medium additives described in the literature for other stem cell culture systems were tried without success. Addition of CEE prepared from homogenized day 7 embryos to the media resulted in a dramatic increase in the percentage of passaged cells that formed colonies and an increase in the rate of colony formation. Although only 5%¨C10% of passaged cells were able to form colonies, this was sufficient to allow expansion of single colonies (Fig. 1E, 1F). For the most part, only type 1 and type 2 colonies passaged efficiently. Colonies could be passaged up to 12 times, after which the rate of colony formation and cell growth diminished.

The Puromycin-Resistant Transgenes Are Integrated
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The transgene bearing the pac cassette is 14 kilobases (kb) long and also contains the coding sequence for human interferon -2B (IFN) (Fig. 2). The pac and IFN sequences are separated by a BamHI site unique to the linearized transgene. Eight unique BDC colonies were cultured until passages 6 to 10, depending on the clone, to obtain a sufficient number of cells for DNA extraction. A probe to the IFN sequence detected bands of various sizes from genomic DNA digested by BamHI in seven of the eight clones (Fig. 3A). The IFN probe detected junction fragments greater than the minimum size expected for an integrated transgene (8.5 kb) in five clones. A fragment smaller than 8.5 kb was detected with clones 2, 5, and 8, suggesting that the transgene had undergone a rearrangement or deletion. A probe to the pac sequence also detected bands in seven of the eight clones (Fig. 3B). Two bands were detected in clone 3. The upper band at 15 kb could be a remnant of the band detected by the IFN probe during the previous hybridization, or the band could be real, revealing either a concatemer fragment or two integration events. Neither the IFN or pac probes detected bands in clone 6 for unknown reasons. It is possible that either or both of the junction fragments were too large to be resolved by standard Southern analysis. The bands detected by both the IFN and pac probes in clones 5 and 8 were very similar in size. Either this was a coincidence, or integration occurred before a BDC divided and formed two colonies in the same original well. Except with the possibility of clone 3, concatemer fragments, which would be 14 kb and revealed by both the IFN and pac probes, were not seen, indicating that the transgene had integrated as a single copy in the majority of events.

Figure 2. Anatomy of a single-copy random insertion. Top line represents the transgene bearing IFN and either the pac or EGFP expression element integrated into a region of a chicken chromosome. The linearized transgene region is denoted by the bracket. The transgene has a single BamHI site between the IFN and pac/EGFP sequences. In this example, the transgene integrated between two genomic BamHI sites that were, prior to integration, 13 kb apart. In a Southern blot analysis of this example, an IFN probe should detect a BamHI-digested fragment of 17.5 kb, and a pac probe should detect a fragment of 9.5 kb. Abbreviations: EGFP, enhanced green fluorescent protein; IFN, interferon; kb, kilobase(s); pac, puromycin resistance gene.
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Figure 3. Transgene integration in puromycin resistance (purr) and enhanced green fluorescent protein-positive (EGFP ) blastodermal cell (BDC)-derived cells. DNA from passaged BDCs was digested by BamHI, and transgenes were identified by Southern blotting and probing with the interferon (IFN) (A) or puromycin resistance gene (pac) (B) coding sequences. (A): Lane 1, nontransgenic BDCs. Lanes 2 to 9, purr, passage 6 BDCs, clones 1 to 8, respectively. Lane 10, an EGFP  clone at passage 11. Lane 11, nontransgenic embryo fibroblasts. White arrows denote bands that correspond to the transgene. The dark band at 12 kb and the lighter bands that are present in all samples were due to nonspecific hybridization of the IFN probe to genomic sequences. The arrow at 8.5 kb indicates the minimum size of a band detected by the IFN probe that would correspond to an intact and integrated transgene. (B): The membrane from (A) was stripped and probed with the pac sequence. White arrows denote bands that correspond to the transgene. The IFN probe that nonspecifically hybridized in (A) was not completely stripped, resulting in the light bands seen in all lanes, including the nontransgenic controls. The arrow at 5.5 kb indicates the minimum size of a band detected by the pac probe that would correspond to an intact and integrated transgene. The dark band at 14.5 kb in lane 4 is likely the same band detected by the IFN probe due to incomplete stripping. (C): Purified blood DNA from putative chimeras was analyzed by quantitative polymerase chain reaction using the 62-base pair neomycin primer/probe set. Five chicks had significant levels of the transgene as indicated by an amplification curve that initiated before cycle 35. Samples that gave rise to amplification curves that initiated after cycle 35 were either negative or had very low levels of the transgene. Abbreviations: Rn, relative increase in fluorescence; kb, kilobase(s); M, molecular weight markers.

Transformation of Blastodermal Cells by Electroporation of a Linearized EGFP Vector

As an alternative to puromycin selection, the use of EGFP as a marker gene for stable integration was investigated. In the absence of puromycin, primary BDCs divide rapidly, such that colonies of several hundred cells form within days of electroporation. The rapid division rate encourages the dilution of episomal DNA, facilitating visual identification of colonies with a stably integrated transgene. Transgene vectors bearing the EGFP coding sequence driven by the RSV promoter were linearized and electroporated into BDCs. With 4¨C8 days of electroporation, BDC colonies were examined for the distribution of EGFP-expressing cells. Colonies with variegated EGFP expression were not picked, as it was likely that the transgene had not integrated in the cells of those colonies (Fig. 4A, 4B). Colonies in which a significant fraction of cells expressed EGFP or that had a sector of cells, all of which expressed EGFP, were picked (Fig. 4C, 4D). For every 1,000,000 electroporated BDCs, approximately three colonies with homogenous EGFP expression could be observed. Colonies were picked and passaged in the presence of CEE-containing media on feeder cells. At passage 1, a number of colonies did not express EGFP and were likely derived from contaminating cells that lacked an integrated transgene. The EGFP-positive colonies were reisolated at passage 1, such that by passage 2 and subsequent passages, all colonies were EGFP-positive (Fig. 4E, 4F).2 E, v* C- D! U9 J' ]6 G
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Figure 4. Isolation and passaging of homogenous enhanced green fluorescent protein-positive (EGFP ) blastodermal cell (BDC) colonies. Four to eight days after electroporation, BDC cultures were imaged for EGFP, and colonies harboring integrated transgenes were selected based on the distribution of cells expressing EGFP in each colony. (A, B): A colony displaying nonhomogenous expression of EGFP, indicating transient expression of EGFP. (C, D): A colony in which one sector is homogenous for EGFP expression, indicating stable integration. (E, F): EGFP  colonies at passage 6. Left panels, UV light image; right panels, visible image. Scale bar = 100 µm.8 o8 H( _4 F. v
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DNA was extracted from one line of EGFP-positive cells at passage 11 to confirm integration by Southern analysis. A single band at 13 kb was detected by the IFN probe, suggesting that the transgene was present and integrated (Fig. 3A, lane 10). There was no pac sequence in the EGFP transgene; thus, the pac probe did not hybridize to any bands specific for the transgene.
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Production of Somatic Chimeras with Transgenic BDC-Derived Lines
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Either individual purr BDC colonies or mixtures of up to 60 purr BDC colonies were passaged. At passage 2, 3, or 4, BDCs were harvested and injected into -irradiated stage X White Leghorn embryos. Contribution of the donor cells to somatic tissues was assessed by the extent of BPR plumage in hatchlings and by detection of the transgene in blood DNA. Thirty-seven percent of hatched chicks (17 of 45) had black feathers derived from purr cells, and the fraction of black feathers was as high as 95%. Non-purr cells were able to generate chimeras with BPR plumage at a rate similar to that of purr cells (23%; 4 of 17).

The contribution of cultured BDCs to blood cells in putative chimeras was assessed by quantitative PCR (qPCR) of the transgene in blood DNA. We had previously found that qPCR of a 62-bp region of the neomycin gene allowed sensitive and accurate detection of transgenes in DNA derived from blood and semen of transgenic chickens . So that the same qPCR assay could be used for analysis of chimeras in this study, the 62-bp sequence was inserted into the 3'-untranslated region of the puromycin selection gene such that expression of the gene was not affected. Twelve chicks had significant levels of the transgene in their blood DNA shortly after hatching (Fig. 3C). The transgene level was tested over a 4¨C6-month period and appeared to diminish slowly, although significant levels were still detected in eight birds at 6 months of age (Table 1). Assuming that each transgenic BDC carries a single copy of the transgene, which is supported by the Southern analysis, the percentage of blood cells positive for the transgene was calculated to range from 0.6%¨C14.9%. The level of transgene did seem to correlate with the extent of BPR plumage, although there were several chicks that had a significant amount of BPR plumage that were negative for the transgene in their blood DNA. The ability of cultured BDCs to populate the precursors to blood cells did not appear to be affected by the number of passages, whether colonies were single or a mixed population or the sex of the recipient embryo. The ability of late-passage EGFP cells to form chimeras was not assessed in this study.$ C! V1 v1 w+ F

Table 1. Contribution of passaged purr blastodermal cells to somatic tissues
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Transgenic BDC-Derived Cells Were Detected in the Germline Tissues of Chimeras

To identify male chimeras that carry transgenically modified germ cells, qPCR was performed on purified sperm DNA using the 62-bp neomycin primer/probe set. Four semen samples were collected from each chimera at 2¨C5-day intervals. Several chimeras gave rise to semen samples with a positive qPCR signal, but that was very low and ranged from 0.5%¨C0.05% positive (data not shown). There were no chimeras in which all four semen samples were positive. Several chimeras had either only one or two positive semen samples. To determine whether samples were giving rise to false positives during DNA processing or qPCR analysis, previously positive semen samples that had been archived by freezing were reextracted and retested. In most cases, positive semen samples retested as positive.6 j- B, X) D+ w5 T" _1 v! i+ F5 U

DISCUSSION

As yet, a chicken embryonic stem cell line has not been created, as evidenced by the lack of transgenic, germline chickens produced by such a method. As an alternative to the creation of a chicken embryonic stem cell line, the strategy of this study was to transform freshly isolated BDCs and enrich for those cells that carry an integrated transgene in a time frame during which a reasonable percentage of BDCs were still able to contribute to the germline. We used the endogenous ability of cells to integrate linearized transgenes into random positions of the genome to produce BDCs bearing an integrated transgene. The transformation efficiency of BDCs was low; approximately 1 purr colony was obtained for every 100,000 harvested cells. A similar frequency was obtained using ubiquitous EGFP expression as a marker. It is estimated that, independent of electroporation protocols, 15% of harvested BDCs actually are able to form colonies, so that the actual transformation frequency was approximately 1 in 15,000 cells, which is similar to the frequencies observed for other types of eukaryotic cells.

The low rate at which transgenes integrate in BDCs effectively prohibited the injection of nonselected or nonscreened cells immediately following electroporation into recipient embryos since so few cells carried an integrated transgene. Chicken blastodermal cells can be cultured for short periods of time (

The ability to passage BDCs was a major hurdle in this project. A number of conditions previously reported were attempted without success. However, when BDCs were cultured at high density (>1,000 cells per mm2), a significantly higher proportion of cells survived at subsequent passages (data not shown). Pain et al. reported multiple passages of BDC-derived cells; however, details of the passage protocol, including cell density, were not reported . Additional BDCs were added to the BDC cultures at 48 hours, suggesting that the initial density was high. We maintained a cell density of 300 cells per mm2 or less during primary culture and subsequent passages to facilitate the formation of distinct purr or EGFP  colonies. We found that in high-density cultures, puromycin, as well as other drugs, was not able to effectively remove nontransgenic BDCs, making the identification of purr colonies impossible. High-density culture of EGFP-electroporated cells prevented the formation of distinct EGFP  colonies due to crowding by nontransgenic cells. Therefore, we sought conditions that promoted the culture of BDCs at relatively low densities.+ Z6 S7 j8 d9 S

Addition of CEE to the culture medium in combination with STO feeder cells drastically increased the success of passaging, allowing cell lines to be established from single colonies. CEE contains a variety of unidentified growth factors and cytokines and has factors that stimulate the growth, survival, and/or differentiation of hematopoetic, fibroblast, and nerve cells, among other cell types . The main benefit of CEE in our hands was a dramatic increase in the survival of blastodermal cells after each passage. However, the number of passages was limited to 12, after which the cells went into senescence and atrophied.
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Passaged purr cells were able to contribute to ectodermal tissues, as evidenced by the appearance of BPR plumage in hatched chicks. Transgenes were detected in blood DNA, suggesting that the passaged cells were also able to contribute to mesodermal tissues, which give rise to blood cells. We were able to detect the transgene in DNA extracted from semen samples, suggesting that some of the cultured BDCs had contributed to the germline. However, the percentage of spermatogonial stem cells that were transgenic was very low, as evidenced by the low and variable transgene level in semen samples from positive roosters. Although the transgene could be reliably detected in a given semen sample, additional samples from the same rooster would test negative. This could in part be caused by cycling of the seminiferous epithelium, which is estimated to occur every 2 weeks in chickens . A semen sample could be negative for the transgene if none of the seminiferous tube sections bearing transgenic cells were releasing sperm at the time of collection, a distinct possibility considering the low percentage of transgene-positive sperm cells. The low and variable presence of the transgene in sperm samples indicated that breeding of the roosters to obtain transgenic offspring could have been logistically difficult.4 a: _& d; ^6 v% Y* x
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Recently, lines of chicken embryonic stem (cES) cells were created that could be cultured indefinitely, transformed with transgenes, and were competent to form chimeras . As an alternative to the ES cell strategy, further efforts to improve the efficiency of de novo isolation of transgenic chicken embryonic cells, including BDCs, could lead to a viable platform for the production of transgenic chickens.8 R  F+ l: z$ D1 o

DISCLOSURES
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A.J.H. and Y.W. own stock in AviGenics. A.J.H., C.F.B., S.A.J., and C.F. each received financial support from AviGenics within the last 2 years." c  z. Z7 H- L- B; K3 h1 Z+ {0 X- x2 j
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ACKNOWLEDGMENTS# C# A+ N. F# [; A- W
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We thank Hallie Goldman, Pramod Sutrave, and Gordon Ibeanu for technical assistance and Dr. Robert Ivarie for advice on this project. This study was funded by Grant 1998010026 from the National Institute of Standards and Technology Advanced Technology Program and was supported in part by research funding from AviGenics to A.J.H.
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