Selection of Genetically Modified Chicken Blastodermal Cells by Magnetic-Activated Cell Sorting1

IMMUNOLOGY AND MOLECULAR BIOLOGY Selection of Genetically Modified Chicken Blastodermal Cells by Magnetic-Activated Cell Sorting1 Q. Wei,*,2 B. A. Croy,* and R. J. Etches† *Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada; and †Origen Therapeutics, 1450 Rollins Road, Burlingame, California 94010 successfully transfected cells. The effectiveness of sorting was assessed using X-gal staining to detect lacZ expression from the pmiwZ plasmid. After 48 h of culture, lacZpositive cells appeared to be enriched 1.4-fold in the MACS selected population. Cells from this enriched pool contributed to extra-embryonic and intra-embryonic tissues of 72-h White Leghorn recipient embryos with a marginal increase in levels of intra-embryonic contribution. Our demonstration that transfected, cultured, and magnetically sorted CBC maintain their ability to contribute to ectodermal and mesodermal lineages of intra-embryonic tissues illustrates the potential value of this technique for introducing genetic modifications into birds.

(Key words: chicken blastodermal cell, lacZ, H-2Kk gene, magnetic-activated cells sorting, transgenic) 2001 Poultry Science 80:1671–1678

INTRODUCTION Transfection introduces exogenous DNA to a certain proportion of target cells. Of these, only a small subset will contain stable integration of the DNA into the host genome. To optimize creation of transgenic birds using transfected blastodermal cells, the small subset of chicken blastoderman cells (CBC) expressing the targeted sequence should be enriched. Among the most common strategies used for enrichment of transfected cells are those making use of antibiotic resistance as the selectable marker. However, previous work using Zeocin-based antibiotic selection showed that no selected CBC were able to contribute to tissues of recipient embryos during development (Q. Wei, unpublished data). Thus, it was necessary to explore selection strategies that do not reduce the capacity of the enriched cells to contribute to somatic and germline tissues. Fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are commonly used positive selection tech-

2001 Poultry Science Association, Inc. Received for publication January 2, 2001. Accepted for publication July 9, 2001. 1 This work was performed in Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada. 2 To whom correspondence should be addressed: wqingxia@hotmail. com.

niques. Cells transfected with constructs that encode fluorescently detectable markers can be sorted by FACS. Speksnijder et al. (1999) sorted lacZ-transfected CBC using fluorescent substrates and obtained enrichment. However, the efficiency of transfection and sorting was quite low. Approximately 5% of the initial CBC collected from fresh blastoderms were recovered after sorting for use as inocula (Speksnijder et al., 1999). Surface labeling with antibodies also permits cell sorting by FACS or MACS, with the latter being a more rapid approach. Magneticactivated cell sorting combines immunoconjugation to link cells to magnetic beads and a magnetic field to separate the selected “positive” cells. Use of supermagnetized microbeads (100 to 150 nm diameter) for the enrichment process yields cells that are not significantly altered in function (Pflueger et al., 1990). Successful incorporation of MACS-sorted blastodermal cells into chimeras was reported by Stunden et al. (1998). In their research, nontransfected CBC were enriched by antibody recognization of surface-expressed, germline specific epitopes. After sorting, viable cells injected into compromised recipient embryos contributed to somatic tissues and to the germline. The frequency of somatic chimerism and the rates

Abbreviation Key: CBC = chicken blastodermal cells; FACS = fluorescence-activated cell sorting; FITC = fluorescein isothiocyanate; MACS = magnetic-activated cell sorting; PI = propidium iodide.

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ABSTRACT The use of chicken blastodermal cells (CBC) in the production of transgenic chickens requires incorporation of the desired stable genetic modification into CBC. With greater proportions of stably transfected blastodermal cells in the embryo inoculum, the frequency of intermediate chimeric birds is greater. Magnetic-activated cell sorting (MACS) was evaluated as a method for enrichment of transfected CBC. This approach requires surface expression of a molecule that can be recognized by an antibody. Chicken blastodermal cells from fertilized Barred Plymouth Rock eggs were coelectroporated with pmiwZ and pMACS Kk and were sorted magnetically by expression of the mouse H-2Kk molecule on the surface of

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MATERIALS AND METHODS Preparation of Plasmid DNA for Transfection Both plasmids pmiwZ, containing the lacZ gene under the control of the chicken β-actin promoter (Suemori et al., 1990), and pMACS Kk (catalog # 130070201),3 containing the H-2Kk gene under the control of the mouse H-2Kk promoter, used in this experiment were transformed into Escherichia coli DH5α competent cells, amplified, and isolated by alkaline lysis as described by Birnboim and Doly (1979).

Isolation and Electroporation of CBC Stage X blastoderms were isolated from fertilized, unincubated Barred Plymouth Rock eggs and dispersed into a single cell suspension as described by Petitte et al. (1990). The cells were washed twice with 5 mL of cytomix buffer (van den Hoff et al., 1992) deficient in ATP and glutathione and were resuspended in cytomix buffer containing 2 mM ATP (catalog # A7437)4 and 5 mM glutathione (catalog # G6529).4 Approximately 60 µL that contained 2.0 to 2.5 × 105 washed CBC were added to a 10 µL mixture of plasmids pmiwZ and pMACS Kk at molar ratios of 1:1, 1:1.4, 1:2, or 1:3 in 0.10-cm gap cuvettes and incubated (room temperature, 10 min) prior to electroporation. Electroporation was performed as described by Zajchowski et al. (2000). Immediately after electroporation, 1 mL of OptiMEM complete medium5 or OptiMEM complete medium (37 C) conditioned by mouse SNL 76/7 fibroblast cells (Speksnijder et al., 1999) was added to the cuvette and then incubated (room temperature, 10 min) before plating. Cells from each cuvette were plated on SNL76/

3

Miltenyi Biotec Inc., Auburn, CA 95603. Sigma Chemical Co., St. Louis, MO 63103. Gibco/BRL, Burlington, ON L7P 1A2.

4 5

7 feeder cells and cultured in OptiMEM complete medium for 24, 48, or 72 h. Samples of cultured cells were stained with X-gal5 (37 C, 3 h) to identify cells expressing the lacZ gene (Wei et al., 2000). To assess viability and ability to proliferate after electroporation, about 2 to 2.5 × 105 CBC were plated on 0.1% gelatin-coated, four-well dish and cultured in SNL 76/7 conditioned medium for 24, 48, or 72 h. As a control, similar numbers of CBC that were not transfected were cultured under the same conditions. After 24 h, transfected and not transfected cells were collected, and total numbers of cells were counted using a hemacytometer.

Assessment of Mouse H-2Kk Gene Expression on CBC by Flow Cytometric Cell Counting Cultured cells were dissociated in trypsin-EDTA4 on ice (Speksnijder et al., 1999), recovered, washed once with 3 mL of cold PBF buffer (PBS5 + 0.1% NaN34 + 1% FBS5), and then left on ice for 40 to 60 min. Approximately 1 × 106 recovered cells in 50 µL cold buffer were then delivered to wells of a 96-well plate that each contained 2 µg fluorescein isothiocyanate (FITC) anti-mouse H-2Kk antibody3 in 50 µL PBF buffer. The cells were then incubated (4 C, 12 h) in darkness. Next, the cells were washed twice and resuspended in 250 µL PBF buffer containing 1/104 volume propidium iodide (PI)4 before flow cytometric analysis. A Coulter威EPICS威Elite ESP FACS was used for cell counting. Parameters were set as follows. A single cell suspension of control cells in the absence of PI was first introduced into the instrument to set PMT4 (red fluorescence detector) voltage so that 1.5 to 3% (G1; Figure 1A) of the control cells showed a red autofluorescence signal, which indicated the percentage of dead cells above the first decade of the log scale. Then a sample of control cells exposed to PI (1:104 dilution) was analyzed, and populations of viable (H2) and dead (G2) cells (Figure 1B) were gated, based on the voltage setting shown in Figure 1A. The PMT2 (green fluorescence detector) voltage was adjusted so that 2 to 3% (B1) of the viable control cells (H2) showed a green autofluorescence signal above the first decade (Figure 1C). Finally, the double-stained (with FITC and PI) CBC that were cotransfected with pmiwZ and pMACS Kk were subjected to analysis. Viable cells with higher fluorescence than control cells were detected (Figure 1D).

MACS Chicken blastodermal cells cotransfected with plasmids pmiwZ and pMACS Kk at the molar ratio of 1:2 and cultured in SNL 76/7 conditioned medium for 24 or 48 h were dissociated in trypsin-EDTA and recovered by centrifugation. Approximately 1 × 107 recovered cells in 320 µL PBE (PBS + 0.5% BSA5 + 5 mM EDTA) were magnetically labeled by incubation with 80 µL MACSelect Kk

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of germline chimerism were comparable to the injection of unsorted cells and to those reported by other researchers (Kagami et al., 1995; Etches et al., 1996; Kino et al., 1997). These results encouraged us to sort transfected CBC using MACS based on surface expression of the foreign gene as the targeted molecule. The mouse H-2Kk molecule was chosen as a transgene that might be highly expressed at the host cell surface and for which vectors and antibody reagents are readily available commercially. Chicken blastodermal cells were cotransfected with the plasmid pmiwZ that contains a lacZ reporter gene and pMACS Kk DNA encoding a truncated mouse H-2Kk molecule on the cell surface. The CBC were MACS enriched on the basis of transient expression of H-2Kk. A high percentage of MACS-enriched CBC containing the lacZ reporter gene is anticipated when appropriate ratios of the lacZ reporter gene and the enrichment marker are used.

MAGNETIC-ACTIVATED CELL SORTING OF BLASTODERMAL CELLS

Microbeads3 for 15 min at 10 C. After adding 1.6 mL of PBE, the cell suspension was loaded onto a MS+/RS+ column,3 which was inserted into a MiniMACS magnetic system3 and rinsed with 4 × 500 µL PBE to elute unbound cells (depleted fraction). The column was removed from the magnet, and the magnetically captured cells (enriched fraction) were flushed out with 1 mL PBE. The number and viability of sorted cells after sorting were determined using a hemacytometer and trypan blue dye exclusion. Recovered CBC from unsorted, enriched, and depleted fractions were plated on a 0.1% gelatin-coated, 96-well culture plate at approximately 2.5 × 104 cells/cm2 and were cultured (conditioned OptiMEM, 37 C, 6% CO2, 18 h) before staining with X-gal.

About 5 µL of cell suspension containing 500 CBC exposed to the transfection procedure and MACS were injected into the center of the subgerminal cavity of recipi-

ent White Leghorn embryos prepared as described by Speksnijder et al. (1999). Injected embryos were then incubated (37.5 C, 50% RH, hourly rotation of 90 degrees). After 72 h, the embryos were recovered and stained with X-gal for 3 h. The number of blue cell foci (one cell focus ranged from approximately one to five cells) in extraand intra-embryonic tissues of each chimeric embryo was determined.

Statistical Analysis The effect of culture period and subsequent MACS processing on the level of transfected CBC incorporation to extra-embryonic and intra-embryonic tissues of chimeric embryos was analyzed by a general linear models procedure (SAS/STAT, 1992). The resulting ANOVA tables from this model evaluated the main effects and their interaction. Tukey multiple comparisons (Tukey, 1949) were used to test differences in main effect. Unless otherwise stated, all comparisons were judged significant at P ≤ 0.05.

RESULTS Assessment of Exogenous β-Galactosidase Activity in Cotransfected CBC Approximately 40 to 50% of transfected, unsorted CBC expressed exogenous β-gal activity after cotransfection with pmiwZ and pMACS Kk at ratios of 1:1, 1:1.4, 1:2, and 1:3 and cultured for 24, 48, or 72 h (Figure 2A). At these three points, no significant differences in transfection efficiency were observed (P > 0.05). For each incubation interval, no significant differences were found among the four different molar ratios of the plasmids (P > 0.05). In all experiments, cell viability at harvest from culture was approximately 50%. Although electroporation delayed attachment of CBC to dishes for 4 to 6 h, by 24 h these cells had formed uniform and compact cell colonies, typical in appearance for undifferentiated cells cultured in conditioned medium (Figure 3A).

Assessment of Expression of Mouse H-2Kk Gene in Cotransfected CBC FIGURE 1. Histogram displaying gate setting for flow cytometric cell counting. Control cells and transfected cells (1 × 106) were prepared for flow cytometric cell counting. Gate setting included the following steps: (A) PMT4 (red fluorescence detector) voltage was set so that 1.9% (G1) of the control cells showed auto red fluorescence signal in the absence of propidium iodide (PI). (B) Two populations of cells (viable (H2) and dead (G2) cells) were identified based on the setting in (A) when control cells incubated with PI (1/104 dilution) were analyzed. (C) The PMT2 (green fluorescence detector) voltage was adjusted so that 2.7% (B1) of the viable control cells (H2) showed a green fluorescence signal above the first decade. (D) Approximately 12% (B2) of the viable transfected cell population produced a higher fluorescence signal than the control population. X-axes represent the relative intensity of PI (A, B) or fluorescein isothiocyanate (FITC) anti-mouse H-2Kk (C, D) fluorescence. Y-axes represent the percentage of chicken blastodermal cells (CBC) with red (A, B) or green (C, D) fluorescence.

Approximately 1.5 to 6.0% of the unsorted transfected CBC produced a greater fluorescence signal than the control population after 24 h culture (Figure 2B). The percentage of “positive” cells in the transfected population that was cultured for 48 h ranged from 6 to 12% (Figure 2B), but at 72 h, expression had dropped to <1.5% (Figure 2B). At 24 and 48 h of culture, the 1:2 molar ratio of pmiwZ: pMACS Kk gave the highest expression within each group (Figure 2B; P < 0.05). Therefore, in all subsequent experiments, CBC were cotransfected at a 1:2 molar ratio of pmiwZ: pMACS Kk and were cultured for 24 or 48 h in SNL 76/7 conditioned medium before MACS processing.

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Injection of Unsorted and Sorted CBC into Recipient Embryos

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between the enriched and depleted fractions (P < 0.001) and between unsorted and depleted fractions (P < 0.05) after 48 h of culture.

Incorporation into Recipient Embryos and Distribution of Cultured Transfected Donor Cells Enriched by MACS

Assessment of MACS Efficiency Viability of MACS sorted CBC was >95%, and the percentage of cells recovered was 80%. After 18 h of culture, cells from the H-2Kk-enriched and -depleted fractions formed colonies similar to the control colonies in Figure 3. Figure 4 illustrates sorting efficiencies. In all cases, 45 to 55% of cells in the unsorted population expressed exogenous β-gal activity. There was no significant difference between any two fractions of unsorted, enriched, and depleted cells after culturing transfected CBC for 24 h (P > 0.05). However, after 48 h, MACS enrichment resulted in an approximately 1.4-fold increase in β-gal expressing cells (P < 0.05). There were also significant differences in expression of exogenous β-galactosidase

DISCUSSION The production of embryos containing blue foci clearly indicated that cotransfected, cultured, and MACS-treated CBC can contribute to a chimeric embryo. The efficiency of cotransfection with 5.75 µg of supercoiled pmiwZ and

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FIGURE 2. Assessment of expression of the lacZ gene or mouse H2Kk gene in chicken blastodermal cells (CBC) cotransfected with pmiwZ and pMACS Kk by X-gal staining or flow cytometric cell counting. Freshly isolated CBC were cotransfected with a mixture of plasmids pmiwZ and pMACS Kk at molar ratios of 1:1, 1:1.4, 1:2, or 1:3 by electroporation. After electroporation, cells (about 2.0 to 2.5 × 105) cultured on SNL 76/7 feeder cells for 24, 48, or 72 h were stained with X-gal at pH 7.4, for 3 h to detect the expression of lacZ gene (A). Cells cultured in conditioned OptiMEM complete medium for 24, 48, or 72 h were stained with fluorescein isothiocyanate-labeled anti-mouse H-2Kk antibody and introduced into the flow cytometer as described in Figure 1 to determine the expression of mouse H-2Kk gene on the surface of transfected CBC. Columns represent the means of percentage of lacZor H-2Kk-positive cells determined in three independent experiments; vertical bars represent the standard error of the mean. a–gLetters indicate means that share significant differences between each other. FITC = fluorescein isothiocyanate.

Magnetic-activated cell sorting treatment did not significantly affect the level of incorporation of transfected donor cells into extra-embryonic tissues of recipient embryos (P > 0.05), and no interaction was observed between MACS treatment and time (P > 0.05) (Table 1). The culture of CBC for 48 h significantly reduced the number of blue foci to 5.0 (n = 90 embryos) in extra-embryonic tissues from 11.3 following culture for 24 h (n = 111 embryos) (Table 1). The effect of culture period and interaction between culture period and MACS treatment were significant factors in incorporation of transfected donor cells into intraembryonic tissues (P > 0.05, Table 1). After a 24-h culture, injection of enriched cells significantly reduced the level of intra-embryonic incorporation (n = 36 embryos, = x 0.03) in comparison to injection of unsorted (n = 37 embryos, x = 0.22, P < 0.05) and depleted (n = 38 embryos, x = 0.29, P < 0.05) cells. However, after a 48-h culture, injection of enriched cells yielded a higher average number of blue foci (n = 28 embryos, x = 0.39) in intra-embryonic tissue than after injection of unsorted (n = 30, x = 0.02, P < 0.05) or depleted (n = 32 embryos, x = 0, P < 0.05) cells. In sum, there was essentially no difference in the level of extra- or intra-embryonic incorporation between injection of unsorted and depleted cell fractions after 24 or 48 h of culture. There was, however, a significant difference in the level of intra-embryonic incorporation between enriched CBC and each of the other test populations (unsorted or depleted) after 24 or 48 h of culture. Unsorted and sorted CBC were more likely to incorporate into extra-embryonic tissues than intra-embryonic tissues. Extra-embryonic blue foci were usually located in the border area (Figure 3D) or concentrated to the area vasculosa (Figure 3E). They were much less frequently near the embryo itself (Figure 3F). At 48 h, donor cells derived from the enriched fraction contributed more frequently to intra-embryonic tissues than unsorted and depleted cells. Enriched cells tended to locate in the brain, somites, and unsegmented paraxial mesoderm (Figures 3G, H, and I). Figure 3G shows extensive incorporation of transfected donor cells into the brain, which was not observed in any of control embryos (Figure 3F).

MAGNETIC-ACTIVATED CELL SORTING OF BLASTODERMAL CELLS

different amount of pMACS Kk is approximately 40 to 50%, which is reasonable when compared to previous observations by Zajchowski et al. (2000). In their research, about 70% transfection efficiency was achieved when 10

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µg of supercoiled pmiwZ was used to electroporate CBC under identical conditions. To obviate contamination of mouse fibroblast cells, which could express the mouse H-2Kk molecule on their

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FIGURE 3. Cotransfected chicken blastodermal cells (CBC) cultured in conditioned medium and distribution of unsorted, depleted or enriched CBC in 72-h chimeric chick embryos. About 2 × 105 CBC electroporated with pmiwZ and pMACS Kk at molar ratio of 1:2 were cultured in conditioned OptiMEM in 6% CO2 at 37 C for 24 h (A), 48 h (B), and 72 h (C). Magnification 200×. After culture for 48 h, transfected CBC before or after magnetic-activated cell sorting (MACS) were injected into recipient blastoderms. The viable embryos were recovered and stained with Xgal. (D), (E), and (F) represent extra-embryonic distribution. Blue foci were usually located to the border area (D) or concentrated in the area vasculosa (E) and were occasionally observed near the embryo itself (F). Magnification 100×. (G), (H), and (I) represent intra-embryonic distribution. (G) An extensive incorporation of transfected donor cells into the brain. (H) Blue foci in somite. Magnification 200×. (I) Blue foci in unsegmented paraxial mesoderm. Magnification 100×.

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TABLE 1. The effect of magnetic-activated cell sorting (MACS) and culture period of chicken blastodermal cells (CBC) on incorporation of transfected cells in extra-embryonic and intra-embryonic tissues of 72-h chick embryos Effect of culture period of transfected CBC Average no. of blue foci1 in extra-embryonic tissue/embryo Effect of MACS Unsorted cells Enriched cells Depleted cells x

24 h3 5

10.59 6.287 16.879 11.34a

48 h4 6

6.33 6.118 2.7510 5.00b

x 8.69 6.20 10.41

Average no. of blue foci in intra-embryonic tissue/embryo2 24 h1

48 h2

5,c

6,f

0.22 0.037,d 0.299,c 0.18

0.02 0.398,e 0.0010,f 0.13

x 0.13 0.19 0.16

Main effect means in a row with no common superscript differ significantly (P < 0.05). Means in a column with no common superscript differ significantly (P < 0.05). 1 One blue foci contains one to five lacZ-positive cells. 2 There is significant interaction between effects of culture period and MACS treatment, therefore the data was analyzed by different time period. 3 Four independent experiments. 4 Three independent experiments. 5 n = 37 embryos. 6 n = 30 embryos. 7 n = 36 embryos. 8 n = 28 embryos. 9 n = 38 embryos. 10 n = 32 embryos. a,b c–f

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FIGURE 4. Assessment of magnetic-activated cell sorting (MACS) efficiency. MACS sorted dissociated chicken blastodermal cells (CBC), which were cotransfected with pmiwZ and pMACS Kk at molar ratio of 1:2 and cultured in conditioned medium for 24 or 48 h. The cells recovered from the unsorted, enriched, and depleted fractions were plated in a 0.1% gelatin-coated, 96-well culture plate at approximately 2.5 × 104 cells per cm2. Cells were cultured in conditioned OptiMEM medium for 18 h and stained with X-gal to assess the sorting efficiency. Columns represent the means of percentage of lacZ-positive cells determined in three independent experiments; vertical bars represent the standard error of the mean. a–At each time point, means with no common letter differ significantly (P < 0.05).

surface, the transfected CBC were cultured in SNL 76/7 conditioned OptiMEM medium and formed uniform and compact cell clumps with typical undifferentiated appearance at 24, 48, or 72 h culture (Figure 3A, B, and C). Etches et al. (1996) found that donor cells cultured for 48 h contributed to somatic tissues and the germline of recipient embryos, and their observations made it promising to produce transgenic germline chimeras with genetically modified CBC cultured in conditioned medium. The expression of the mouse H-2Kk gene on CBC was assessed by flow cytometric analysis. There was a significant reduction in expression of H-2Kk at 72 h. The expression profile of the H-2Kk surface marker correlates with the growth rate of cells, with highest expression level occurring roughly after one cell division. The cell cycle of CBC is believed to require about 24 h if manipulated cells behave normally. Therefore, cells cultured for 24 h would have the highest fluorescence signal. However, because electroporation delayed the attachment of CBC to the substrate for about 4 to 6 h, the high level of expression that was detected in transfected cells cultured for 36 to 48 h is consistent with expectation. After 72 h of culture, the level of expression decreased dramatically. Recovery of CBC after MACS processing was 80%, which is superior to that of FACS. The published FACS procedure resulted in the loss of many CBC, leaving only 5% for injection (Speksnijder et al., 1999). The MACS procedure did not compromise the viability of sorted CBC as illustrated by trypan blue staining (<5% dead cells). Sorted CBC were able to attach to plates and form colonies after culture in conditioned medium for 18 h. Unlike FACS, which reduced the rate of proliferation (Spesknijder et al., 1999), cell growth was not decreased following MACS. Therefore, MACS appears to be less detrimental than FACS for sorting CBC.

MAGNETIC-ACTIVATED CELL SORTING OF BLASTODERMAL CELLS

to increase the chances of producing a transgenic chimera if a more efficiently expressed cell surface marker can be found.

ACKNOWLEDGMENTS We thank Kim Brand, Mary Ellen Clark, Janice Brazolot, E. Sasaki, and R. Roberts in Department of Animal and Poultry Science, University of Guelph, Guelph, ON N1G 2W1, for their technical support and helpful discussions. We also thank Margaret Quinton (Department of Animal and Poultry Science, University of Guelph) for her assistance with the statistical analysis. This work was supported by Natural Sciences and Engineering Research Council of Canada, Ontario Ministry of Agriculture, Food and Rural Affairs, and the Human Frontier Science Program.

REFERENCES Birnboim, H. C., and J. Doly, 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513–1523. Brazolot, C. L., J. N. Petitte, R. J. Etches, and A. M. Verrinder Gibbins, 1991. Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo. Mol. Reprod. Dev. 30:304–312. Carsience R. S., M. E. Clark, Verrinder A. M. Gibbins, and R. J. Etches, 1993. Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development 117:669–675. Etches, R. J., M. E. Clark, A, Toner, G. Liu, and A. M. Verrinder Gibbins, 1996. Contribution to somatic and germline lineages of chicken blastodermal cells maintained in culture. Mol. Reprod. Dev. 45:291–298. Fraser, R. A., R. S. Carsience, M. E. Clark, R. J. Etches, and A. M. Verrinder Gibbins, 1993. Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos. Int. J. Dev. Biol. 37:381–385. Kagami, H., M. E. Clark, Verrinder A. M. Gibbins, and R. J. Etches, 1995. Sexual differentiation of chimeric chickens containing ZZ and ZW cell in the germline. Mol. Reprod. Dev. 42:379–487. Kino, K., B. Pain, S. P. Leibo, M. Cochran, and R. J. Etches, 1997. Production of chicken chimeras from frozen-thawed blastodermal cells. Poultry Sci. 76:753–760. Petitte, J. N., M. E. Clark, G. Liu, Verrinder A. M. Gibbins, and R. J. Etches, 1990. Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 108:185–189. Pflueger, E., E. A. Mueller, and F. A. Anderer, 1990. Preservation of cytotoxic function during multi-cycle immunomagnetic cell separations of human NK cells using a new type of magnetic bead. J. Immunol. Methods 129:165–173. SAS/STAT, 1992. Statistical Analysis Systems, User’s Guide. SAS Institute Inc., Cary, NC. Speksnijder, G. J., R. J. Etches, and Verrinder A. M. Gibbins, 1999. Germline chimeric chickens from FACS-sorted donor cells. Mol. Reprod. Dev. 52:33–42. Stunden, C. E., K. Brand, and R. J. Etches, 1998. Enrichment for blastodermal cells expressing germline specific epitopes using magnetic activated cell sorting. Poultry Sci. 77(Suppl. 1):77. (Abstr.). Suemori, H., Y. Kadodawa, K. Goto, I. Araki, H. Kondoh, and N. Nakatsuji, 1990. A mouse embryonic stem cell line showing

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It had been expected that isolation of positive cells with MACS would improve the level of incorporation of transfected donor cells into the recipient embryos. However, at 24 h, the injection of enriched cells decreased the frequency of intra-embryonic chimerism in comparison to the injection of unsorted and depleted cells. One possible explanation is that cotransfected CBC were not able to recover from electrical impairment within 24 h of culture and were still fragile. Microbead binding and buffer flushing of enriched cells during MACS could increase this detrimental effect, which could reduce the ability of enriched CBC to compete with the fast growing intraembryonic cells in the recipient. In contrast, at 48 h, the frequency of intra-embryonic chimerism of birds injected with enriched cells was significantly increased. This result suggests that cotransfected CBC were recovered from electroporation after extended period in culture and could tolerate the small detrimental effect of MACS. Therefore, the enriched CBC were able to contribute to fast growing intra-embryonic tissues in the recipient. The efficiency of producing germline chimeras is related to the efficiency of producing somatic chimeras (Carsience et al., 1993). Therefore, it seems reasonable to assume that increasing the numbers of transfected donor cells that are incorporated into intra-embryonic tissues will also increase the likelihood that a small proportion of germline progenitor cells present in the pooled donor cell population will become incorporated and, therefore, increase the chance of obtaining a germline chimera. The expression of the mouse H-2Kk gene (<12%) in cotransfected CBC was much lower than that of lacZ gene (40 to 50%). Possibly, the efficiency of transgene expression on surface of transfected CBC could be improved using the β-actin promoter to drive H-2Kk because this promoter controlling the lacZ gene in pmiwZ conferred high levels of constitutive expression in chicken cells (Brazolot et al., 1991; Fraser et al., 1993; Speksnijder et al., 1999). Therefore, a new plasmid pmiw-H-2Kk, which contains the mouse H-2Kk gene under the control of the chicken β-actin promoter, was constructed (Q. Wei, unpublished). This vector was functional as it improved the efficiency of H-2Kk expression in transfected CHO cells. However, no expression was detected in CBC transfected with this new pmiw-H-2Kk vector. The inconsistent expression suggests that the mechanisms regulating surface expression of the H-2Kk gene are very complex and may depend on the combined effect of the promoter, enhancer, coding sequences, cell types, and extracellular environment. In conclusion, the mouse H-2Kk antigen can be expressed on the surface of CBC cotransfected with pmiwZ and pMACS Kk. By using this selectable surface marker, transfected CBC cultured for 48 h can be enriched by MACS, and injection of the enriched cells marginally increases levels of intra-embryonic donor cell incorporation in 72-h recipient embryos. Furthermore, the transfected, cultured, and sorted cells maintain their ability to contribute to ectodermal and mesodermal lineages. These results suggest that MACS might be a practical method by which

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pluripotency of differentiation in early embryos and ubiquitous β-galactosidase expression. Cell Differ. Dev. 29:181–186. Tukey, J. W., 1949. Comparing individual means in the analysis of variance. Biometrics 5:99–114. van den Hoff, M. J. B., A. F. M. Moorman, and W. H. Lamers, 1992. Electroporation in “intracellular” buffer increases cell survival. Nucleic Acids Res. 20:2902.

Wei., Q., W. L. Woods, and R. J. Etches, 2000. Long-term culture of chicken blastodermal cells (CBCS) and selection of transfected cells using antibiotic resistance. Methods Mol. Biol. 136:399–403. Zajchowski, L. D., S. M. Mohammed, Q. Wei, and R. J. Etches, 2000. Incorporation of genetically modified cells in chicken chimeras. Methods Mol. Biol. 136:391–397.

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