Development 108, 185-189(1990)Printed in Great Britain © The Company of Biologists Limited 1990

185

Production of somatic and germline chimeras in the chicken by transfer of

early blastodermal cells

J. N. PETITTE, M. E. CLARK, G. LIU, A. M. VERRINDER GIBBINS and R. J. ETCHES*

Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada NIC 2W1

•To whom correspondence and reprint requests should be addressed

Summary

Cells were isolated from stage X embryos of a line ofBarred Plymouth Rock chickens (that have black pig-ment in their feathers due to the recessive allele at the Ilocus) and injected into the subgerminal cavity ofembryos from an inbred line of Dwarf White Leghorns(that have white feathers due to the dominant allele atthe I locus). Of 53 Dwarf White Leghorn embryos thatwere injected with Barred Plymouth Rock blastodermalcells, 6 (11.3 %) were phenotypically chimeric withrespect to feather colour and one (a male) survived tohatching. The distribution of black feathers in therecipients was variable and not limited to a particularregion although, in all but one case, the donor celllineage was evident in the head. The male somaticchimera was mated to several Barred Plymouth Rockhens to determine the extent to which donor cells hadbeen incorporated into his testes. Of 719 chicks hatchedfrom these matings, 2 were phenotypically Barred Ply-mouth Rocks demonstrating that cells capable of incor-

poration into the germline had been transferred. Finger-prints of the blood and sperm DNA from the germlinechimera indicated that both of these tissues were differ-ent from those of the inbred line of Dwarf WhiteLeghorns. Bands that were present in fingerprints ofblood DNA from the chimera and not present in those ofthe Dwarf White Leghorns were observed in those of theBarred Plymouth Rocks. It was concluded that cellsrecovered from the stage X embryo can subsequentlycontribute to melanocytes derived from the neural crest,to erythrocytes and to germ cells. This technique ofblastodermal cell transfer should be useful in develop-mental studies and may facilitate the production oftransgenic poultry either directly or through the estab-lishment of chicken pluripotent stem cell lines in vitro.

Key words: blastoderm, chick, chimera, germline,melanocytes.

Introduction

During the past several years, the production of trans-genic animals has been a prominent theme in animalbiology. In the mouse, foreign DNA has been intro-duced into the genome by microinjection of newlyfertilized eggs (Palmiter et al. 1982), through infectionusing retroviral vectors (Jaenisch, 1976) and followingincorporation of DNA into embryonic stem cells(Gossler et al. 1986; Robertson et al. 1986). In thechicken, microinjection into newly fertilized eggs isdifficult because the ovum is coated with a mucin-richmembrane within 15 min after ovulation, is sub-sequently surrounded by several grams of albumen andan eggshell, and is fragile when collected from theupper regions of the oviduct (Rowlett and Simkiss,1987; Perry, 1988). However, foreign DNA has beeninserted into the chicken germline by injecting eitherreplication-competent avian leukosis viruses (Salter etal. 1987) or replication-defective reticuloendothelialviruses (Bosselman et al. 1989) into the embryo before

incubation. At this time, the blastoderm contains30000-40000 cells, is believed to be pluripotent (Eyal-Giladi, 1984), and will accept transplanted tissue (Mar-zullo, 1970). We have demonstrated that isolated cellsfrom the stage X embryo can be transferred from oneembryo to another. These transferred cells will enterthe germline, at least one derivative of neural crest cells(i.e. the melanocytes) and the hemopoietic tissues. Thismethod of producing germline chimeras may be usefulin the development of transgenic chickens.

Materials and methods

Donor blastoderms were obtained from an inbred line ofBarred Plymouth Rock chickens that were homozygousrecessive (ii) at the dominant white locus. Freshly oviposited,unincubated eggs were cracked open and the blastodermsremoved by adherence to filter paper rings (Lucas andJamroz, 1961) and placed in a Petri dish with 20 ml of Medium199 (pH7.2) supplemented with 4.3mM-NaHCO3, gentamy-cin (20/igmr1) or penicillin (lOOi.u. mr')/streptomycin

186 /. N. Petitte and others

(lOOjjgml '). The yolk was removed by microdissection andgentle washing with medium. Only stage X (Eyal-Giladi andKochav, 1976) blastoderms were used. Intact blastodermswere dissected free from the vitelline membrane, placed inlml of fresh medium and washed twice to remove anyremaining yolk. Cell dissociation was accomplished by replac-ing the medium with 1 ml of 0.25 % trypsin/0.04 % EDTA(w/v) in phosphate-buffered saline and incubation at 37 °C for10 min. The cells were dispersed by repeated aspiration of themedium into a Pasteur pipet. After dissociation, the cells werecentrifuged and washed with 1 ml of medium containing 20 %fetal bovine serum (v/v). A sample of cell suspension wasused to determine viability (>90 %, by trypan blue exclusion)and concentration. Prior to injection, the cells were resus-pended in 0.1-0.2 ml of medium.

Recipient eggs were obtained from an inbred line of DwarfWhite Leghorn chickens that were homozygous dominant (II)at the dominant white locus. Freshly laid eggs were swabbedwith 70% alcohol and a 0.5 cm window was made in theequatorial plane of the eggshell directly over the blastoderm.Approximately 200-500 cells were injected into the subgermi-nal cavity in 2-5 /<1 of medium using a finely drawn micro-pipet. The windows were sealed with paraffin wax and a glasscoverslip or with an adhesive film permeable to gases andwater vapour (Opsite, Smith & Nephew, Quebec). The eggswere candled about every seven days and dead embryos wereexamined for evidence of chimerism. Control eggs werewindowed but not injected or were fresh, non-windowed eggs.

All chicks that hatched were classified as putative chimerasif they were phenotypically Dwarf White Leghorn or somaticchimeras if some of their feathering was pigmented. Thosechicks that survived to sexual maturity were mated to BarredPlymouth Rocks to test for germline chimerism. To verify thephenotype of the crossbred chick, reciprocal matings betweenDwarf White Leghorns and Barred Plymouth Rocks weremade.

Detection of the descendents of the donor cells that hadincorporated into blood and semen was achieved by finger-print analysis. DNA was isolated from whole blood (25 /d)according to the method of Signer et al. (1988) followed bythree extractions with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, by volume) and dialysisagainst buffer solution (50mM-Tris-HCl, 100mM-NaCl, 2mM-EDTA,pH8.0). For the isolation of DNA from semen, 100(Aof semen was mixed with 1 ml of a solution containing 10 ITIM-Tris-HCl, lmM-EDTA at pH8.0, and incubated at 37°C for30 min. Next, 20fd of 2-mercaptoethanol and 25^1 of 20%(w/v) sodium dodecyl sulphate solution were added and themixture incubated at 50 °C for 30 min. Following addition ofRNase A (25 /il of a 20mgml~1 solution) and incubation at50°C for 30 min, proteinase K (30^1 of a 20 mg ml"1 solution)was added and incubation continued at 50°C for 2 h and 37°Covernight. The addition of NaCl and subsequent steps were

performed as for the isolation of DNA from blood. IsolatedDNA was digested to completion with Pall [Pharmacia(Canada) Inc., Dorval, Quebec], according to the supplier'sinstructions, and resulting fragments were subjected to elec-trophoresis through 0.9% (w/v) agarose gels for 48 h at1 volt cm"' using 89mM-Tris-HCl, 89mM-boric acid, lmM-EDTA (pH8.3) buffer. DNA in the gel was transferred toGeneScreen Plus membrane (DuPont Co., Boston, Mass.) byvacuum blotting using IOXSSC (1.5M-NaCl, 0.15M-sodiumcitrate, pH7.0). The resulting blot was probed with M13mpl8 double-stranded DNA (Vassart et al. 1987) labelledwith [32P]dCTP (SOOOCimmor1,10/zCi /il"1; ICN Biochemi-cals Canada Ltd, Montreal, Quebec) using the RandomPrimers Labeling System (GIBCO/BRL, Burlington,Ontario). Hybridization conditions were according to West-neat et al. (1988).

Results

Of the 53 Dwarf White Leghorn embryos that wereinjected with dissociated blastodermal cells fromBarred Plymouth Rock embryos, four survived tohatch. The hatchability of the injected eggs was notsignificantly different from that of the uninjected win-dowed eggs (Table 1, ^=0.3847, P>0.l0) but wassignificantly lower than that observed for the non-windowed eggs 0^=55.4, P<0.001). The low hatch-ability was not associated with the injection procedureper se, but appears to be due to the windowingprocedure. The exact reason for this decline in hatcha-bility is unknown although it has been reported pre-viously (Marzullo, 1970).

Six of the injected embryos were phenotypicallychimeric as indicated by the presence of black feathers;one chimera survived to hatch and was a male(Fig. 1A). Among the embryos that died during incu-bation, the extent of the black pigmentation variedconsiderably from only a small spot to nearly 90 % ofthe feather tracts (Fig. 1B-D). In all but one case ofphenotypic chimerism, black feathers were observed inthe head region. With each successive moult, thechimeric male exhibited less pigmentation in his headuntil he was phenotypically identical to a Dwarf WhiteLeghorn in his adult plumage (Fig. 2A). Of the fourchicks that hatched, the somatic chimera shown inFig. 1A and two putative female chimeras were raisedto sexual maturity and mated to Barred PlymouthRocks.

Matings between the somatically chimeric male and

Table 1. Embryonic mortality and survival to hatching following transfer of blastodermal cells from stage XBarred Plymouth Rock embryos into Dwarf White Leghorns at the same stage of development

Treatment

Barred Plymouth Rock toDwarf White Leghorn

Uninjected Controls:WindowedNo Window

No.1, Number of embryos.

No.1

53

2223

<8

52.8

40.94.3

% Mortality at

10-16

18.8

31.80

day

17-20

20.7

18.20

% Hatched

7.5

9.195.6

Fig. 1. (A) A somatic and germline chimera which hatched after injection of dissociated blastodermal cells from BarredPlymouth Rock embryos into the subgerminal cavity of a Dwarf White Leghorn embryo. This chick (a male) was raised tosexual maturity and is shown in his adult plumage in Fig. 2A. (B,C and D) Embryos exhibiting somatic chimerism after 14days of incubation. The most extreme incorporation of the Barred Plymouth Rock cell line is shown in B where most of thefeather tracts are black. Intermediate levels of incorporation are shown in C and the lowest level of incorporation is shownin D. In most cases, the donor cell line is incorporated into melanocytes around the head.

Fig. 2. (A) The somatic chimeric male in his adult plumage. Note that the pigmentation which was evident in this bird athatch was not present at sexual maturity. (B) A typical Barred Plymouth Rock hen in adult plumage. (C) A BarredPlymouth Rock chick and 3 Barred Plymouth RockxDwarf White Leghorn chicks that resulted from mating the chimera inA with Ban-ed Plymouth Rock hens as shown in B. (D) The Barred Plymouth Rock that was sired by the chimera andshown as a chick in C at 14 weeks of age. The fingerprint of blood DNA from this chick is shown in Fig. 3 A,B. lane 11,

1 2 3 4 5 6 7 8 9 10 11 12 13

1Somatic and germline chimeras in chickens 187

2 3 4 5 6 7 8 9 10 11 12 13

!J-f1"

Fig. 3. DNA fingerprints. Blood and sperm DNA were digested to completion with Pad, subjected to electrophoresisthrough 0.9% agarose gels for 48 h at 1 volt cm"1, vacuum blotted onto GeneScreen Plus membrane, and probed with M13mpl8 DNA labelled with pPjdCTP by the random primer method. The resulting autoradiographs are shown: (A) lanes 1-6and 8, blood from individual Dwarf White Leghorns; lanes 7 and 12, blood from individual Barred Plymouth Rocks; lane 9,chimera semen; lane 10, chimera blood; lane 11, blood from the first chick sired by the chimera that was phenotypicallyBarred Plymouth Rock; lane 13, blood from the second chick sired by the chimera that was phenotypically Barred PlymouthRock. (B) lanes 1-8 and 12, blood from individual Barred Plymouth Rocks; lanes 9-11 and 13 as for (A).

Barred Plymouth Rock hens (shown in Fig. 2B) pro-duced 2 Barred Plymouth Rock chicks and 717 chicksexhibiting the yellow with occasional black fleckingphenotype characteristic of a Dwarf White Leghorn xBarred Plymouth Rock cross (Fig. 2C, D). The twoputative chimeric hens produced 24 and 5 chicks,respectively, of the Dwarf White Leghorn x BarredPlymouth Rock phenotype.

Fingerprints of blood DNA from several birds fromthe highly inbred line of Dwarf White Leghorn that wasused to provide recipients were very similar to eachother (Fig. 3A, lanes 1-6, 8) whereas those of theBarred Plymouth Rocks were variable (Fig. 3B, lanes1-8, 12). Fingerprints of semen and blood DNA fromthe chimera revealed that only minor differencesexisted in the DNA from these two tissues (Fig. 3A and3B, lanes 9 and 10) and that blood from the chimeracontained DNA that was different from that found inthe Dwarf White Leghorns. The bands that werepresent in fingerprints of blood DNA from the chimerabut not seen in those of the Dwarf White Leghorns wereevident in fingerprints of blood DNA from the BarredPlymouth Rocks. Fingerprints of blood DNA from thechimera offspring with the Barred Plymouth Rockphenotype contained bands that were typically found in

BarTed Plymouth Rock fingerprints and not present inthose of the Dwarf White Leghorn blood DNA.

Discussion

The technique described in this report has consistentlyyielded somatic cell chimerism in approximately 10 %of all recipient embryos in our laboratory during thepast 12 months. By transferring clumps of cells fromembryos that were unstaged but obtained from unincu-bated eggs, Marzullo (1970) was able to produce 3phenotypically chimeric embryos from 239 recipients,although none of these chicks survived to hatching. Ourtechnique, therefore, is a considerably more successfulmethod and this improvement is most likely due to theuse of dissociated cells and only stage X blastoderms(Eyal-Giladi and Kochav, 1976) as both donors andrecipients. The stage X blastoderm is probably bestsuited to the formation of chimeras because the areapellucida of the stage X embryo is composed of a singlelayer of epiblast cells that subsequently give rise to allembryonic tissues (Vakaet, 1962). By stage XII, thehypoblast, which induces the formation of the primitivestreak (Eyal-Giladi, 1984; Khaner and Eyal-Giladi,1989; Eyal-Giladi and Khaner, 1989), has started to

188 J. N. Petitte and others

differentiate suggesting that cells taken from this stageof embryonic development are less likely to be pluripo-tent.

A complete analysis of the contribution of the trans-ferred blastodermal cells to all of the somatic tissues inthe chimeras has not yet been accomplished. However,it is evident from the black pigmentation of the feathersin the chimeras during late embryonic development andat hatching that Barred Plymouth Rock blastodermalcells contributed to the melanocytes which are derivedfrom neural crest cells. In addition, the fingerprints ofblood DNA indicate that these blastodermal cellscontribute to the progenitors of the erythrocytes. It isimpossible, however, to determine from these resultswhether somatic chimerism is due to the introductionand subsequent integration of committed cells or due tothe presence of pluripotential cells in the donor.

The fingerprints of semen DNA and the breedingrecord of the chimera provide evidence that the transferof stage X blastodermal cells can result in germlinechimerism. Germline chimerism may have developedfrom the injection of pluripotential cells or from theintroduction of cells that were previously committed todifferentiate into primordial germ cells (PGCs). In thechick, PGCs have been shown to arise from the epiblast(Eyal-Giladi et al. 1981; Urven et al. 1988) and to beginmigration to the developing hypoblast at stage XII(Sutasurya et al. 1983; Urven et al. 1988). Duringgastrulation, the PGCs continue to migrate via thehypoblast and mesoderm into an area called the germi-nal crescent (Swift, 1914; Ginsburg and Eyal-Giladi,1986). Subsequently, with the formation of the extra-embryonic vasculature (stages 12-13 of Hamburger andHamilton, 1951), PGCs can be found in embryonicblood samples up to stage 20 when they begin to settleinto the gonadal ridge (Singh and Meyer, 1967; Swartzand Domm, 1972; Ando and Fujimoto, 1983). Byexamining the in vitro differentiation of PGCs invarious fragments of the stage X blastoderm, Ginsburgand Eyal-Giladi (1987) demonstrated that PGCs orig-inate from the central disc of the area pellucida. Thetotal number of PGCs per fragmented embryo wassimilar to that of control embryos suggesting that thecells that are destined to become PGCs may already bedetermined by stage X. Further studies are required toexamine the interactions between the donor cells andthe recipient embryo that lead to germline chimerism.

Somatic and germline chimeras developed by transferof dissociated stage X blastodermal cells have manypotential applications in developmental studies usingthe chick embryo as a model vertebrate system. Forexample, it is unclear why the melanophores in thechimera were functional at hatching but did not pig-ment the adult plumage. This unique model may beuseful in the study of the way in which cells interactduring pigmentation. Germline chimeras may also be auseful vehicle in the development of transgenic chick-ens if techniques for the establishment of avian embry-onic stem cells can be found. Assuming that mouse andchicken germline chimeras are analogous, it should bepossible to create transgenic chickens using a similar

strategy to that described by Bradley et al. (1984) for themouse. Development of these techniques would facili-tate the use of homologous recombination and site-directed mutagenesis in studies where manipulation ofthe chicken genome was either the goal in itself or ameans of introducing specific changes in gene ex-pression in order to study the biology of embryonicdevelopment.

The authors wish to acknowledge the technical assistance ofMrs Gertraude Hurnik and the staff at the Arkell PoultryResearch Station. This work was supported by grants from theNatural Sciences and Engineering Research Council, theOntario Ministry of Agriculture and Food, and the OntarioEgg Producers' Marketing Board. We thank Drs M. H.Fallding and A. L. Joyner for their helpful suggestions in thepreparation of this manuscript.

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(Accepted 9 October 1989)