Cells were isolated from stage X embryos of a line of Barred Plymouth Rock chickens (that have black pigment in their feathers due to the recessive allele at the I locus) and injected into the subgerminal cavity of embryos from an inbred line of Dwarf White Leghorns (that have white feathers due to the dominant allele at the I locus). Of 53 Dwarf White Leghorn embryos that were injected with Barred Plymouth Rock blastodermal cells, 6 (11.3 %) were phenotypically chimeric with respect to feather colour and one (a male) survived to hatching. The distribution of black feathers in the recipients was variable and not limited to a particular region although, in all but one case, the donor cell lineage was evident in the head. The male somatic chimera was mated to several Barred Plymouth Rock hens to determine the extent to which donor cells had been incorporated into his testes. Of 719 chicks hatched from these matings, 2 were phenotypically Barred Plymouth Rocks demonstrating that cells capable of incorporation into the germline had been transferred. Fingerprints of the blood and sperm DNA from the germline chimera indicated that both of these tissues were different from those of the inbred line of Dwarf White Leghorns. Bands that were present in fingerprints of blood DNA from the chimera and not present in those of the Dwarf White Leghorns were observed in those of the Barred Plymouth Rocks. It was concluded that cells recovered from the stage X embryo can subsequently contribute to melanocytes derived from the neural crest, to erythrocytes and to germ cells. This technique of blastodermal cell transfer should be useful in developmental studies and may facilitate the production of transgenic poultry either directly or through the establishment of chicken pluripotent stem cell lines in vitro.

During the past several years, the production of transgenic animals has been a prominent theme in animal biology. In the mouse, foreign DNA has been introduced into the genome by microinjection of newly fertilized eggs (Palmiter et al. 1982), through infection using retroviral vectors (Jaenisch, 1976) and following incorporation of DNA into embryonic stem cells (Gossler et al. 1986; Robertson et al. 1986). In the chicken, microinjection into newly fertilized eggs is difficult because the ovum is coated with a mucin-rich membrane within 15 min after ovulation, is subsequently surrounded by several grams of albumen and an eggshell, and is fragile when collected from the upper regions of the oviduct (Rowlett and Simkiss, 1987; Perry, 1988). However, foreign DNA has been inserted into the chicken germline by injecting either replication-competent avian leukosis viruses (Salter et al. 1987) or replication-defective reticuloendothelial viruses (Bosselman et al. 1989) into the embryo before incubation. At this time, the blastoderm contains 30000–40000 cells, is believed to be pluripotent (Eyal-Giladi, 1984), and will accept transplanted tissue (Mar-zullo, 1970). We have demonstrated that isolated cells from the stage X embryo can be transferred from one embryo to another. These transferred cells will enter the germline, at least one derivative of neural crest cells (i.e. the melanocytes) and the hemopoietic tissues. This method of producing germline chimeras may be useful in the development of transgenic chickens.

Donor blastoderms were obtained from an inbred line of Barred Plymouth Rock chickens that were homozygous recessive (ii) at the dominant white locus. Freshly oviposited, unincubated eggs were cracked open and the blastoderms removed by adherence to filter paper rings (Lucas and Jamroz, 1961) and placed in a Petri dish with 20 ml of Medium 199 (pH 7.2) supplemented with 4.3 mM-NaHCO3, gentamy-cin (20/μgmr1) or penicillin (100i.u. ml−1)/streptomycin (100μgml−1). The yolk was removed by microdissection and gentle washing with medium. Only stage X (Eyal-Giladi and Kochav, 1976) blastoderms were used. Intact blastoderms were dissected free from the vitelline membrane, placed in 1ml of fresh medium and washed twice to remove any remaining yolk. Cell dissociation was accomplished by replacing the medium with 1ml of 0.25% trypsin/0.04 % EDTA (w/v) in phosphate-buffered saline and incubation at 37 °C for 10 min. The cells were dispersed by repeated aspiration of the medium into a Pasteur pipet. After dissociation, the cells were centrifuged and washed with 1 ml of medium containing 20 % fetal bovine serum (v/v). A sample of cell suspension was used to determine viability (>90 %, by trypan blue exclusion) and concentration. Prior to injection, the cells were resuspended in 0.1–0.2 ml of medium.

Recipient eggs were obtained from an inbred line of Dwarf White Leghorn chickens that were homozygous dominant (II) at the dominant white locus. Freshly laid eggs were swabbed with 70% alcohol and a 0.5 cm window was made in the equatorial plane of the eggshell directly over the blastoderm. Approximately 200–500 cells were injected into the subgerminal cavity in 2–5 μl of medium using a finely drawn micropipet. The windows were sealed with paraffin wax and a glass coverslip or with an adhesive film permeable to gases and water vapour (Opsite, Smith & Nephew, Quebec). The eggs were candled about every seven days and dead embryos were examined for evidence of chimerism. Control eggs were windowed but not injected or were fresh, non-windowed eggs.

All chicks that hatched were classified as putative chimeras if they were phenotypically Dwarf White Leghorn or somatic chimeras if some of their feathering was pigmented. Those chicks that survived to sexual maturity were mated to Barred Plymouth Rocks to test for germline chimerism. To verify the phenotype of the crossbred chick, reciprocal matings between Dwarf White Leghorns and Barred Plymouth Rocks were made.

Detection of the descendents of the donor cells that had incorporated into blood and semen was achieved by fingerprint analysis. DNA was isolated from whole blood (25 μl) according to the method of Signer et al. (1988) followed by three extractions with an equal volume of phenol/ chloroform/isoamyl alcohol (25:24:1, by volume) and dialysis against buffer solution (50 mM-Tris-HCl, 100 mM-NaCl, 2 mM-EDTA, pH 8.0). For the isolation of DNA from semen, 100/d of semen was mixed with 1 ml of a solution containing 10 mM-Tris-HCl, ImM-EDTA at pH8.0, and incubated at 37°C for 30 min. Next, 20 μ1 of 2-mercaptoethanol and 25 μl of 20% (w/v) sodium dodecyl sulphate solution were added and the mixture incubated at 50°C for 30 min. Following addition of RNase A (25 μI of a 20 mg ml−1 solution) and incubation at 50°C for 30 min, proteinase K (30 μl of a 20 mg ml−1 solution) was added and incubation continued at 50°C for 2 h and 37°C overnight. The addition of NaCl and subsequent steps were performed as for the isolation of DNA from blood. Isolated DNA was digested to completion with Pall [Pharmacia (Canada) Inc., Dorval, Quebec], according to the supplier’s instructions, and resulting fragments were subjected to electrophoresis through 0.9% (w/v) agarose gels for 48 h at 1 volt cm−1 using 89 mM-Tris-HCl, 89mM-boric acid, ImM-EDTA (pH8.3) buffer. DNA in the gel was transferred to GeneScreen Plus membrane (DuPont Co., Boston, Mass.) by vacuum blotting using lOxSSC (1.5M-NaCl, 0.15M-sodium citrate, pH7.0). The resulting blot was probed with M13 mpl8 double-stranded DNA (Vassart et al. 1987) labelled with [32P]dCTP (3000 Ci mmol−1,10 μCi μl−1; ICN Biochemicals Canada Ltd, Montreal, Quebec) using the Random Primers Labeling System (GIBCO/BRL, Burlington, Ontario). Hybridization conditions were according to Westneat et al. (1988).

Of the 53 Dwarf White Leghorn embryos that were injected with dissociated blastodermal cells from Barred Plymouth Rock embryos, four survived to hatch. The hatchability of the injected eggs was not significantly different from that of the uninjected windowed eggs (Table 1, χ2=0.3847, P>0.10) but was significantly lower than that observed for the nonwindowed eggs (χ2=55.4, PcO.001). The low hatchability was not associated with the injection procedure per se, but appears to be due to the windowing procedure. The exact reason for this decline in hatchability is unknown although it has been reported previously (Marzullo, 1970).

Table 1.

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

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

Six of the injected embryos were phenotypically chimeric 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 incubation, the extent of the black pigmentation varied considerably from only a small spot to nearly 90 % of the feather tracts (Fig. 1BD). In all but one case of phenotypic chimerism, black feathers were observed in the head region. With each successive moult, the chimeric male exhibited less pigmentation in his head until he was phenotypically identical to a Dwarf White Leghorn in his adult plumage (Fig. 2A). Of the four chicks that hatched, the somatic chimera shown in Fig. 1A and two putative female chimeras were raised to sexual maturity and mated to Barred Plymouth Rocks.

Fig. 1.

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

Fig. 1.

(A) A somatic and germline chimera which hatched after injection of dissociated blastodermal cells from Barred Plymouth Rock embryos into the subgerminal cavity of a Dwarf White Leghorn embryo. This chick (a male) was raised to sexual maturity and is shown in his adult plumage in Fig. 2A. (B,C and D) Embryos exhibiting somatic chimerism after 14 days of incubation. The most extreme incorporation of the Barred Plymouth Rock cell line is shown in B where most of the feather tracts are black. Intermediate levels of incorporation are shown in C and the lowest level of incorporation is shown in 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 at hatch was not present at sexual maturity. (B) A typical Barred Plymouth Rock hen in adult plumage. (C) A Barred Plymouth Rock chick and 3 Barred Plymouth Rock×Dwarf White Leghorn chicks that resulted from mating the chimera in A with Barred Plymouth Rock hens as shown in B. (D) The Barred Plymouth Rock that was sired by the chimera and shown 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.

Fig. 2.

(A) The somatic chimeric male in his adult plumage. Note that the pigmentation which was evident in this bird at hatch was not present at sexual maturity. (B) A typical Barred Plymouth Rock hen in adult plumage. (C) A Barred Plymouth Rock chick and 3 Barred Plymouth Rock×Dwarf White Leghorn chicks that resulted from mating the chimera in A with Barred Plymouth Rock hens as shown in B. (D) The Barred Plymouth Rock that was sired by the chimera and shown 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.

Matings between the somatically chimeric male and Barred Plymouth Rock hens (shown in Fig. 2B) produced 2 Barred Plymouth Rock chicks and 717 chicks exhibiting the yellow with occasional black flecking phenotype characteristic of a Dwarf White Leghorn × Barred Plymouth Rock cross (Fig. 2C, D). The two putative chimeric hens produced 24 and 5 chicks, respectively, of the Dwarf White Leghorn×Barred Plymouth Rock phenotype.

Fingerprints of blood DNA from several birds from the highly inbred line of Dwarf White Leghorn that was used to provide recipients were very similar to each other (Fig. 3A, lanes 1–6, 8) whereas those of the Barred Plymouth Rocks were variable (Fig. 3B, lanes 1–8, 12). Fingerprints of semen and blood DNA from the chimera revealed that only minor differences existed in the DNA from these two tissues (Fig. 3A and 3B, lanes 9 and 10) and that blood from the chimera contained DNA that was different from that found in the Dwarf White Leghorns. The bands that were present in fingerprints of blood DNA from the chimera but not seen in those of the Dwarf White Leghorns were evident in fingerprints of blood DNA from the Barred Plymouth Rocks. Fingerprints of blood DNA from the chimera offspring with the Barred Plymouth Rock phenotype contained bands that were typically found in Barred Plymouth Rock fingerprints and not present in those of the Dwarf White Leghorn blood DNA.

Fig. 3.

DNA fingerprints. Blood and sperm DNA were digested to completion with Pah, subjected to electrophoresis through 0.9% agarose gels for 48 h at 1 volt cm−1, vacuum blotted onto GeneScreen Plus membrane, and probed with M13 mpl8 DNA labelled with [32P]dCTP by the random primer method. The resulting autoradiographs are shown: (A) lanes 1–6 and 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 phenotypically Barred Plymouth Rock; lane 13, blood from the second chick sired by the chimera that was phenotypically Barred Plymouth Rock. (B) lanes 1–8 and 12, blood from individual Barred Plymouth Rocks; lanes 9–11 and 13 as for (A).

Fig. 3.

DNA fingerprints. Blood and sperm DNA were digested to completion with Pah, subjected to electrophoresis through 0.9% agarose gels for 48 h at 1 volt cm−1, vacuum blotted onto GeneScreen Plus membrane, and probed with M13 mpl8 DNA labelled with [32P]dCTP by the random primer method. The resulting autoradiographs are shown: (A) lanes 1–6 and 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 phenotypically Barred Plymouth Rock; lane 13, blood from the second chick sired by the chimera that was phenotypically Barred Plymouth Rock. (B) lanes 1–8 and 12, blood from individual Barred Plymouth Rocks; lanes 9–11 and 13 as for (A).

The technique described in this report has consistently yielded somatic cell chimerism in approximately 10% of all recipient embryos in our laboratory during the past 12 months. By transferring clumps of cells from embryos that were unstaged but obtained from unincubated eggs, Marzullo (1970) was able to produce 3 phenotypically chimeric embryos from 239 recipients, although none of these chicks survived to hatching. Our technique, therefore, is a considerably more successful method and this improvement is most likely due to the use of dissociated cells and only stage X blastoderms (Eyal-Giladi and Kochav, 1976) as both donors and recipients. The stage X blastoderm is probably best suited to the formation of chimeras because the area pellucida of the stage X embryo is composed of a single layer of epiblast cells that subsequently give rise to all embryonic tissues (Vakaet, 1962). By stage XII, the hypoblast, which induces the formation of the primitive streak (Eyal-Giladi, 1984; Khaner and Eyal-Giladi, 1989; Eyal-Giladi and Khaner, 1989), has started to differentiate suggesting that cells taken from this stage of embryonic development are less likely to be pluripotent.

A complete analysis of the contribution of the transferred blastodermal cells to all of the somatic tissues in the chimeras has not yet been accomplished. However, it is evident from the black pigmentation of the feathers in the chimeras during late embryonic development and at hatching that Barred Plymouth Rock blastodermal cells contributed to the melanocytes which are derived from neural crest cells. In addition, the fingerprints of blood DNA indicate that these blastodermal cells contribute to the progenitors of the erythrocytes. It is impossible, however, to determine from these results whether somatic chimerism is due to the introduction and subsequent integration of committed cells or due to the presence of pluripotential cells in the donor.

The fingerprints of semen DNA and the breeding record of the chimera provide evidence that the transfer of stage X blastodermal cells can result in germline chimerism. Germline chimerism may have developed from the injection of pluripotential cells or from the introduction of cells that were previously committed to differentiate into primordial germ cells (PGCs). In the chick, PGCs have been shown to arise from the epiblast (Eyal-Giladi et al. 1981; Urven et al. 1988) and to begin migration to the developing hypoblast at stage XII (Sutasurya et al. 1983; Urven et al. 1988). During gastrulation, the PGCs continue to migrate via the hypoblast and mesoderm into an area called the germinal crescent (Swift, 1914; Ginsburg and Eyal-Giladi, 1986). Subsequently, with the formation of the extra-embryonic vasculature (stages 12–13 of Hamburger and Hamilton, 1951), PGCs can be found in embryonic blood samples up to stage 20 when they begin to settle into the gonadal ridge (Singh and Meyer, 1967; Swartz and Domm, 1972; Ando and Fujimoto, 1983). By examining the in vitro differentiation of PGCs in various fragments of the stage X blastoderm, Ginsburg and Eyal-Giladi (1987) demonstrated that PGCs originate from the central disc of the area pellucida. The total number of PGCs per fragmented embryo was similar to that of control embryos suggesting that the cells that are destined to become PGCs may already be determined by stage X. Further studies are required to examine the interactions between the donor cells and the recipient embryo that lead to germline chimerism.

Somatic and germline chimeras developed by transfer of dissociated stage X blastodermal cells have many potential applications in developmental studies using the chick embryo as a model vertebrate system. For example, it is unclear why the melanophores in the chimera were functional at hatching but did not pigment the adult plumage. This unique model may be useful in the study of the way in which cells interact during pigmentation. Germline chimeras may also be a useful vehicle in the development of transgenic chickens if techniques for the establishment of avian embryonic stem cells can be found. Assuming that mouse and chicken germline chimeras are analogous, it should be possible to create transgenic chickens using a similar strategy to that described by Bradley et al. (1984) for the mouse. Development of these techniques would facilitate the use of homologous recombination and site-directed mutagenesis in studies where manipulation of the chicken genome was either the goal in itself or a means of introducing specific changes in gene expression in order to study the biology of embryonic development.

The authors wish to acknowledge the technical assistance of Mrs Gertraude Humik and the staff at the Arkell Poultry Research Station. This work was supported by grants from the Natural Sciences and Engineering Research Council, the Ontario Ministry of Agriculture and Food, and the Ontario Egg Producers’ Marketing Board. We thank Drs M. H. Fallding and A. L. Joyner for their helpful suggestions in the preparation of this manuscript.

Ando
,
Y.
and
Fujimoto
,
T.
(
1983
).
Ultrastructural evidence that primordial cells leave the blood-vascular system prior to migration to the gonadal analgen
.
Dev. Growth Diff
.
25
,
345
352
.
Bosselman
,
R. A.
,
Hsu
,
R.
,
Boggs
,
T.
,
Hu
,
S.
,
Bruszewski
,
J.
,
Ou
,
S.
,
Kozar
,
L.
,
Martin
,
F.
,
Green
,
C.
,
Jacobsen
,
F.
,
Nicholson
,
M.
,
Schulz
,
J. A.
,
Semon
,
K. M.
,
Richell
,
W.
and
Stewart
,
R. G.
(
1989
).
Germline transmission of exogenous genes in the chicken
.
Science
243
,
533
535
.
Bradley
,
A.
,
Evans
,
M.
,
Kaufman
,
H. M.
and
Robertson
,
E.
(
1984
).
Formation of germ-line chimeras from embryo-derived teratocarcinoma cell lines
.
Nature, Land
.
309
,
255
256
.
Eyal-Giladi
,
H.
(
1984
).
The gradual establishment of cell commitments during the early stages of chick development
.
Cell Diff
.
14
,
245
255
.
Eyal-Giladi
,
H.
,
Ginsburg
,
M.
and
Farbarov
,
M.
(
1981
).
Avian primordial cells are of epiblastic origin
.
J. Embryol. exp. Morph
.
65
,
139
147
.
Eyal-Giladi
,
H.
and
Khaner
,
O.
(
1989
).
The chick’s marginal zone and primitive streak formation II. Quantification of the marginal zone’s potencies - temporal and spatial aspects
.
Devi Biol
.
134
,
215
221
.
Eyal-Giladi
,
H.
and
Kochav
,
S.
(
1976
).
From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of development of the chick
.
Devi Biol
.
49
,
321
337
.
Ginsburg
,
M.
and
Eyal-Giladi
,
H.
(
1986
).
Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo
.
J. Embryol. exp. Morph
.
95
,
53
71
.
Ginsburg
,
M.
and
Eyal-Giladi
,
H.
(
1987
).
Primordial germ cells of the young chick blastoderm originate from the central zone of the area pellucida irrespective of the embryo-forming process
.
Development
101
,
209
219
.
Gossler
,
A.
,
Doetschman
,
T.
,
Korn
,
R.
,
Serfling
,
E.
and
Kemler
,
R.
(
1986
).
Transgenesis by means of blastocyst-derived embryonic stem cell lines
.
Proc. natn. Acad. Sci. U. S. A
.
83
,
9065
9069
.
Hamburger
,
V.
and
Hamilton
,
H. L.
(
1951
).
A series of normal stages in the development of the chick
.
J. Morph
.
88
,
49
92
.
Jaenisch
,
R.
(
1976
).
Germ line integration and mendelian transmission of the exogenous moloney leukemia virus
.
Proc, natn. Acad. Sci. U. S. A
.
73
,
1260
1264
.
Khaner
,
O.
and
Eyal-Giladi
,
H.
(
1989
).
The chick’s marginal zone and primitive streak formation I. Coordinativo effect of induction and inhibition
.
Devi Biol
.
134
,
206
214
.
Lucas
,
A. M.
and
Jamroz
,
C.
(
1961
).
Atlas of Avian Hematology, U.S. Department of Agriculture, Washington, D.C., p.225
.
Marzullo
,
G.
(
1970
).
Production of chick chimaeras
.
Nature, Lond
.
225
,
72
73
.
Palmiter
,
R. D.
,
Brinster
,
R. L.
,
Hammer
,
R. E.
,
Trumbauer
,
M. E.
,
Rosenfeld
,
M. G.
,
Birnberg
,
N. C.
and
Evans
,
R. M.
(
1982
).
Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes
.
Nature, Land
.
300
,
611
615
.
Perry
,
M. M.
(
1988
).
A complete culture system for the chick embryo
.
Nature, Lond
.
331
,
70
72
.
Robertson
,
E.
,
Bradley
,
A.
,
Kuehn
,
M.
and
Evans
,
M.
(
1986
).
Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector
.
Nature, Lond
.
323
,
445
448
.
Rowlett
,
K.
and
Simkiss
,
K.
(
1987
).
Explanted embryo culture: in vitro and in ovo techniques for domestic fowl
.
Br. Poult. Sci
.
28
,
91
101
.
Salter
,
D.W.
,
Smith
,
E.J.
,
Hughes
,
S.H.
,
Wright
,
S.E.
and
Crittenden
,
L.B.
(
1987
).
Transgenic chickens: insertion of retroviral genes into the chicken germ line
.
Virology
157
,
236
240
.
Signer
,
E.
,
Kuenzle
,
C. C.
,
Thomann
,
P. E.
and
Hubscher
,
U.
(
1988
).
DNA fingerprinting: improved DNA extraction from small blood samples
.
Nucleic Acids Res
.
16
,
7738
.
Singh
,
R. P.
and
Meyer
,
D. B.
(
1967
).
Primordial germ cells in blood smears from chick embryos
.
Science
156
,
1503
1504
.
Sutasurya
,
L. A.
,
Yasugi
,
S.
and
Mizuno
,
T.
(
1983
).
Appearance of primordial germ cells in young chick blastoderms cultured in vitro
.
Develop. Growth Differ
.
25
,
517
521
.
Swartz
,
W. J.
and
Domm
,
L. V.
(
1972
).
A study on the division of primordial germ cells in the early chick embryo
.
Am. J. Anar
.
135
,
51
70
.
Swift
,
C. H.
(
1914
).
Origin and early history of the primordial germ-cells in the chick
.
Am. J. Anat
.
15
,
483
516
.
Urven
,
L. E.
,
Erickson
,
C. A.
,
Abbot
,
U. K.
and
McCarrey
,
J. R.
(
1988
).
Analysis of germ fine development in the chick embryo using an antimouse EC cell antibody
.
Development
103
,
299
304
.
Vakaet
,
L.
(
1962
).
Some new data concerning the formation of the definitive endoblast in the chick embryo
.
J. Embryol. exp. Morph
.
10
,
38
56
.
Vassart
,
G.
,
Georges
,
M.
,
Monsieur
,
R.
,
Brocas
,
H.
,
Lequarre
,
A. S.
and
Christophe
,
D.
(
1987
).
A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA
.
Science
235
,
683
684
.
Westneat
,
D. F.
,
Noon
,
W. A.
,
Reeve
,
H. K.
and
Aquadro
,
C. F.
(
1988
).
Improved hybridization conditions for DNA “fingerprints” probed with M13
.
Nucleic Acids Res
.
16
,
4161
.