A Modified Method of Shell Windowing for Producing Somatic or ...

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Research Note A Modified Method of Shell Windowing for Producing Somatic or Germline Chimeras in Fertilized Chicken Eggs G. Speksnijder1 and R. Ivarie2 Department of Genetics, University of Georgia, Athens, Georgia 30602-7223 ABSTRACT Stage X chick blastoderms following oviposition were accessed via a small window in the egg. Windowing, however, substantially reduces the hatchability of eggs containing early embryos. For example, only 32 of 389 (8.2%) eggs hatched after standard windowing with or without irradiation or injection. Ex ovo culture systems can overcome this problem but are labor intensive. A modification of a standard windowing technique has yielded an average hatch rate of 32% for 892 windowed eggs independent of incubator type, γ-irradiation, or injection of the embryo. This was a fourfold increase over a standard windowing method. Similar hatch rates were observed using fertile eggs from five chicken lines [Barred Plymouth Rock (BR), Athens-Canadian

(AC), Line 0, SPAFAS, and commercial White Leghorns (WL)]. The modification involves covering the egg shell membrane with PBS after grinding away the shell and before piercing the membrane. The window is then sealed by overlaying with fresh shell membrane and cementing it in place once it has dried. The method has been used successfully for the production of somatic and germline chimeras because donor BR blastodermal cells injected into Stage X, γ-irradiated recipient embryos from WL or AC yielded a hatch of 33.7%, of which 42.3% were feather chimeras. Two of 11 cockerels tested were germline mosaics bearing at least 1% BR sperm. The modified windowing technique may be broadly applicable in emerging technologies in avian transgenesis and development.

(Key words: chicken, window, chimera, transgenic, blastogerm) 2000 Poultry Science 79:1430–1433

INTRODUCTION Access to the early avian embryo is important for the production of transgenic birds. Whether modification is achieved by introduction of genetic material at the singlecell stage or by introducing vectors of foreign DNA at the multicellular stage, physical manipulation of the embryo is required. In chickens, transgenic modification of the genome was achieved by microinjecting DNA into fertilized zygotes, but the procedure required sacrificing the donor hen by surgical removal of the oocyte from the magnum of the oviduct (Love et al., 1994). Subsequent development of the embryo to hatch also required an elaborate ex ovo culture system (Rowlett and Simkiss, 1987; Perry, 1988; Naito et al., 1990) or reintroduction of the embryo with attached yolk into a recipient hen by means of a fistula (Pancer et al., 1989). Although transgenic birds have been obtained successfully via these approaches, they are labor intensive, inefficient, and require large donor flocks.

Received for publication November 15, 1999. Accepted for publication June 19, 2000. 1 Current address: AviGenics, Inc., 220 Riverbend Rd., Athens, GA 30602. 2 To whom correspondence should be addressed: ivarie@arches. uga.edu.

The chicken embryo is most easily accessed following oviposition. The embryo, a blastoderm at Stage X (EyalGiladi and Kochav, 1976; hereafter referred to as EG-K), can be accessed by opening a hole or “window” in the shell. Embryo manipulation at this stage can involve direct or indirect genetic modification of the blastoderm via retroviral transducing vectors (Bosselman et al., 1989; Thoraval et al., 1995) or via injection of genetically modified donor cells into its subgerminal cavity (Brazolot et al., 1991; Fraser et al., 1993). Cutting a window in the eggshell, however, results in high incidence of embryonic defects, often leading to death, especially if the egg is windowed at early embryonic stages (Fisher and Schoenwolf, 1983). The windowing process exposes the egg contents to the atmosphere, allowing entry of air. Resealing the egg with this artificial air cell is apparently detrimental to the embryo because filling this air space with liquid before closing the window can rescue the embryo, at least partially, from the teratogenic effects of windowing (Fisher and Schoenwolf, 1983; Fineman et al., 1986; Fineman and Schoenwolf, 1987). Here, a simple modification of a standard windowing technique is described that allows physical manipulation

Abbreviation Key: AC = Athens-Canadian, BR = Barred Plymouth Rock, EG-K = Eyal-Giladi and Kochav, PBS-G = PBS containing 5.6 mM D-glucose, WL = White Leghorn.

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of the blastoderm that results in an acceptable hatch rate independent of incubator type with relatively little operator dependence. The method can be used to produce somatic and germline chimeric chickens efficiently and enable the production of transgenic birds.

MATERIALS AND METHODS Isolation of Donor Blastodermal Cells Blastodermal cells were collected from Stage X (EGK) embryos as described by Petitte et al. (1990). Freshly oviposited eggs were cracked, freed of egg white, and placed into Petri dishes with the blastoderm facing up (3 to 4 yolks per 100-mm dish). A filter paper ring (Whatman #1,3 inside diameter approximately 7 mm) was placed around the blastoderm, and the vitelline membrane was cut around the outside of the ring. The ring with the adhering blastoderm was removed from the yolk and placed ventral side up in sterile PBS containing 5.6 mM D-glucose (PBS-G4). Yolk was removed from the blastoderm by microdissection and gentle rinsing with PBS-G, and the area pellucida was separated from the area opaca of the embryo with a hair loop. The area pellucidae were then transferred to fresh PBS-G and allowed to settle for 10 min on ice. The cells were rinsed once in calcium- and magnesium-free PBS4 and allowed to settle for 10 min on ice before incubating for 10 min on ice in 0.05% trypsin (wt/vol)/0.53 mM EDTA in calcium- and magnesiumfree PBS. This solution was aspirated and replaced with Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum, and the cells were vortexed briefly at low speed and centrifuged at 570 × g for 5 min at room temperature. The cells were resuspended in Dulbecco’s Modified Eagle’s Medium plus 10% fetal bovine serum and kept on ice during injections into recipient embryos.

turning the egg under illumination using a fiber optic light source (Fiber Lite model 1807). Only blastoderms at or near Stage X (EG-K) were injected. Using a drawn 50µL micropipette (50 to 60 µm outer diameter), approximately 5 µL of cell suspension (500 to 1000 cells) were injected into the subgerminal cavity of the recipient blastoderm. The window was sealed with an approximately 1-cm square of fresh shell membrane soaked in PBS. Fresh shell membrane was removed from egg shell fragments with forceps just before each experiment. Orientation of the membrane was the same as it was in the egg. The patch was dried before covering with Duco cement. Eggs were incubated narrow end down at 37.5 C/60% relative humidity and were turned 90° each hour for 18 d. Eggs were then placed in hatching baskets and incubated at 36.9 C/70% relative humidity until hatch. This procedure was modified as follows. A small volume of PBS (e.g., PBS-G without glucose) was placed on the shell hole prior to piercing the shell membrane so that a droplet formed above and around the hole. The membrane was removed, allowing PBS, not air, to be drawn into the egg interior. Some eggs obtained commercially (e.g., SPAFAS) absorbed the PBS during manipulation and required additional PBS. Also, commercial eggs that had been cleaned did not hold the PBS droplet well. The latter problem was solved by smearing a small amount of petroleum jelly around the hole prior to the addition of PBS.

Statistical Analysis Statistical significance of the results was evaluated by the z statistic, which measures differences in proportions (Dixon and Massey, 1983). The control hatch rate from the standard windowing experiment without irradiation and injection was used to calculate z values in Table 1 for White Leghorn (WL) and Barred Plymouth Rock (BR).

Windowing and Injecting Eggs In the standard windowing procedure, fresh, fertile eggs were exposed to 500 rd of γ-irradiation from a 60Co source (Carsience et al., 1993), swabbed with 70% ethanol, and placed horizontal with respect to their long axis for several hours. A hole approximately 5 mm in diameter was ground through the shell at the top of the horizontal egg using a Dremel moto-tool (Model 7505) fitted with an aluminum oxide grinding wheel (Mounted Norton Wheel 75115696). Care was taken not to breach the underlying shell membrane (Bosselman et al., 1989). After swabbing the newly ground hole with 70% ethanol, the shell membrane was cut cleanly around the hole with a #11 scalpel blade to remove the membrane without leaving jagged edges. The underlying blastoderm was located by

3

Whatman Int. Ltd., Maidstone, UK. Gibco BRL, Grand Island, NY 14702. 5 Dremel Mfg., Racine, WI 53406. 6 Manhattan System Co., Mapleton, GA 30126. 7 Dolan Jenner Industries, Lawrence, MA 01843. 4

RESULTS AND DISCUSSION Hatchability of eggs windowed using the standard technique described in Materials and Methods was compared with that using our modification of adding PBS above the hole in the shell prior to cutting the underlying shell membrane. Table 1 is a summary of experiments using eggs from WL, BR, or Athens-Canadian (AC) strains of chickens. Similar results have also been obtained with Line 0 and SPAFAS strains (data not shown). The first column indicates whether the standard or modified windowing technique was used. Adjacent columns indicate whether eggs were irradiated prior to windowing and whether they were injected. Irradiation has been shown to increase the frequency of feather chimerism in part by delaying the development of the recipient embryo (Carsience et al., 1993). White Leghorn and AC eggs containing Stage X embryos were injected with freshly collected and dispersed BR blastodermal cells (Stage X), and BR eggs were injected with WL blastodermal cells. The standard windowing technique resulted in hatch rates

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SPEKSNIJDER AND IVARIE TABLE 1. Hatchability of windowed eggs Windowing conditions Strain

STD1

New

Irradiated

Injected

Eggs set

Eggs hatched

Percent hatched

N2

White Leghorn

+ + + − − − − + − − − −

− − − + + + + − + + + +

− − + − + − + − − + + +

− + + − − + + − − − + +

69 70 201 51 73 73 334 49 18 68 203 72

6 4 19 18 26 22 108 3 2 22 62 29

8.7 5.7 9.5 ± 2.9 35.3 35.6 30.1 32.3 ± 2.9 6.1 11.1 32.4 30.5 ± 1.8 40.3

1 2 6 1 1 2 10 1 1 1 5 2

Barred Plymouth Rock

Athens-Canadian

z value3 −0.68 0.19 3.60 3.84 3.21 3.97 0.69 3.42 3.51 ND4

1

Standard windowing method. Number of trials for each set of conditions; standard errors are given for those experiments with >2 trials. 3 Hatch rates of unirradiated, uninjected control embryos for standard windowing were used for each chicken line in calculating the z value. 4 Not determined. 2

ranging from 6 to 9%, regardless of whether the eggs were irradiated, injected, or irradiated and injected. These results confirm earlier reports of the effect of windowing on hatchability (Petitte et al., 1999; Thoraval et al., 1995). The addition of PBS prior to piercing the shell membrane, however, produced a significant increase in hatch rates. Hatchability of eggs windowed with the new technique averaged over 30% and exceeded 50% in two experiments. Statistical significance was determined by the z statistic for differences in proportions (Dixon and Massey, 1983). For WL and BR embryos, the control hatch rate via standard windows without irradiation or injection was used as the value against which the new method and further manipulations were tested. In general, z values greater than or equal to 1.96 are statistically significant at P < 0.05. Using the new windowing method, the lowest value of z, with one exception, was 3.21 with P = 0.0014. However, only 2 of 18 hatched in one trial for unknown reasons (z = 0.69). Further manipulations to windowing, such as irradiation or injection, seemed to have no compromising effect on embryo hatchability. The hatch rates would also have been considerably higher if corrected for the hatch frequency of control, unwindowed eggs. However, the results were not normalized for hatchability because the windowed eggs were selected for healthy

Stage X embryos, and nonfertile or unhealthy embryos were discarded. Finally, hatchability was compared in different incubators (Jamesway AVN, NatureForm NOM125, and Jamesway 252), but no incubator dependence was observed. Upon hatching, WL and AC chicks from embryos injected with BR cells were scored for somatic feather chimerism from donor melanocytes. Donor embryos were obtained from inter se matings of BR that were homozygous recessive at the I locus (i/i) causing black feather pigmentation. Recipient embryos from inter se matings of WL or AC were homozygous dominant at the I locus (I/I) and, therefore, had white feathers. Chimeric chicks with melanocytes derived from injected donor cells, therefore, should show black feathering. Table 2 shows that the modified windowing technique had no deleterious effect on somatic chimerism, eliminating the need for ex ovo culture of injected embryos after 3 to 4 d by transfer to surrogate shells (Petitte et al., 1990; Pain et al., 1996), thereby greatly reducing the labor required to produce chimeric birds. Large numbers of somatic chimeras, therefore, can be more easily produced which, in turn, allows more somatic chimeras to be tested for germline transmission.

TABLE 2. Chimerism of injected embryos Windowing conditions Strain

STD

New

Irradiated

Injected

Eggs set

Eggs hatched (%)

Somatic chimeras (%)

Germline chimeras2

N3

White Leghorn

+ + − − −

− − + + +

− + − + +

− + + + +

70 201 73 334 72

4 19 22 108 29

2 7 11 50 8

0/2 2/3 0/3 2/9 0/2

2 6 2 10 2

Athens-Canadian 1

1

(5.7) (9.5) (30.1) (32.3) (40.3)

(50.0) (36.8) (50.0) (46.3) (27.6)

Standard windowing method. Number of males that were germline chimeras versus the number of males tested. 3 Number of trials for each set of conditions. 2

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To test for germline transmission, chimeric male chicks were raised to sexual maturity and then bred to BR hens. Barred Rock chicks resulting from these crosses indicate that some of the injected donor cells formed functional germ cells in the recipient embryo. At least 200 offspring were bred from each male. As shown in Table 2, approximately 1% of sperm from 2 of 11 male chimeras produced by injection of BR cells into γ-irradiated embryos bore the i allele, indicating that the new windowing technique yields transmission of a donor cell trait. The modified windowing technique described in this report allows manipulation of the Stage X (EG-K) embryo and its subsequent development to hatch, thereby yielding viable and sexually mature birds. By contrast, standard windowing techniques severely impaired chick development. Besides BR, WL, and AC, the method has produced similar hatch rates for fertile eggs bearing Stage X (EG-K) embryos from SPAFAS and Line 0 birds (data not shown). The reason for the rescuing effect of the new technique is unknown, but it is proposed that introduction of an artificial air space over the blastoderm causes mechanical stress on the embryo (Fineman and Schoenwolf, 1987). Although difficult to measure, stress placed on the blastoderm during the windowing procedure seems to produce a range of early embryonic defects that often lead to death. For reasons as yet unclear, eliminating the introduction of air during the windowing procedure allows proper development and hatch of a large percentage of embryos. It is likely that other aqueous solutions could be used in the modified method in place of PBS, such as thin albumen, saline solution, water, or culture medium. The simple modification to the windowing method described here allows access to the early chick embryo. The addition of steps toward transgenic modification of the embryo may allow this method to be used for production of transgenic chickens. The method can be used to introduce genetically modified blastodermal cells to produce transgenic chimeras or it can be used to introduce any of a number of DNA vectors directly into the embryo. The higher rate of hatch suggests fewer injections of donor cells or vectors may be necessary to identify transgenic chimeras.

ACKNOWLEDGMENTS The authors thank Alex Harvey and Mike McDonell (AviGenics, Inc., Athens, GA 30602) for their helpful comments on the manuscript and Daniel Promislow in the Genetics Department at The University of Georgia (Athens, GA 30602) for help with the statistical analysis. On campus, we also thank Henry Marks and Bill Burke in the Poultry Science Department for use of the poultry facilities for parts of this work and John Noakes in the Center for Applied Isotope Studies for the use of the 60 Co source. This work was supported from grants from

Technology Development Program of the Georgia Research Alliance and AviGenics, Inc.

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