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Production
of chicken chimeras by fusing blastodermal cells with electroporation
S. Aritomi, N. Fujihara
Division
of Animal Resource Science, Faculty of Agriculture, Kyushu University
Graduate
School, Fukuoka 812-8581, Japan
Asian J Androl 2000 Dec; 2: 271-275
Keywords:
chicken
blastoderm; electroporation; chimera
Abstract
Aim: To establish techniques for producing somatic and germline chimeric chicken by transferring blastodermal cells fused with electroporation. Methods: Stage-X blastodermal cells isolated from freshly laid fertile unincubated white Leghorn and Rhode Island red chicken eggs were fused with electroporation. The treated cell suspension was transferred to the recovery medium (DMEM containing 10% FBS) and was injected into the subgerminal cavity of recipient unincubated embryos (stage X). Results: Of 177 recipient embryos injected with the fusing blastodermal cells, 6 (3.4%) survived to hatching. Somatic chimerism was examined in the melanocyte of the feather. The presence of feathers originating from the donor cell was observed in 1 bird (16.7%) out of the 6 hatched birds. After 21 days of incubation two birds out of five embryos were subjected to polymerase chain reaction (PCR) analysis for W-chromosome-specific DNA for each tissue. One bird possessed W-chromosome-specific DNA in the stomach, and the other exhibited the same DNA in the left and right gonads and other tissues, but not the stomach. Conclusion: Recipient embryo having electrofused blastodermal cells yields somatic and germline chimeric chickens more successfully.
1 Introduction
The introduction of foreign DNA into chicken has gained increasing popularity as a method for the production of transgenic or chimeric chicken, in a hope of facilitating chicken breeding. Recently, two important methods have been developed. One is the technique of foreign DNA microinjection into the undifferentiated fertilized ovum, and the other is electroporation with a high reproducibility and easy manipulation[1,2]. The principle of electroporation is the application of one or several short and sufficiently strong electric pulses to cell suspensions or monolayer, so that, some parts of the cell membrane will be broken down temporarily with the formation of permeable minute pores. The surrounding chemicals such as DNA or other materials may diffuse or be driven into the cells during the time when the cell membrane becomes permeable[1]. The advantage of electroloading is that the process is applicable to almost all cell types, ranging from bacteria to human tissues. The other technique is the injection of primordial germ cells (PGCs)[3,4] or dispersed blastodermal cells from Stage X eggs[5-8]. Stage X embryos[9,10] are very useful for producing chimeric chickens, as at this stage, the embryo is composed of 40,000 to 80,000 morphologically undifferentiated cells. In the previous study, a lot of somatic and germ-line chimeric chickens have been produced by treating recipient eggs. One method is the irradiation of recipient embryos prior to injection of the donor cells at stage X[11-14]. The other is the injection of cytotoxic drug busulphan to destroy migrating primordial germ cells[15]. However, these methods have problems that the rate of germ-line chimera production is very low. Therefore, we tried to produce somatic-and germline chimeric chickens by transferring two-strain blastodermal cells fused with electroporation method. This method of producing germline chimeras may be useful for the development of transgenic chickens in the future. In the present experiments we tried to create chickens having testis-like organs which will also produce spermatozoa carrying W-chromosome specific gene by transgenesis.2 Materials and methods
2.1
Cell fusion with electroporation
Eggs
were obtained from white Leghorn and Rhode Island red hens and were artificially
inseminated. Water-soluble polymers used in the two-phase polymer systems
were polyethylene glycol (PEG, MW 8,000, SIGMA) and dextran (Dx, MW 7,000,
TCI)[1]. For the experiments and the confirmation of gene transfer,
pEGFP (green fluorescence protein)-N1 Vector ( 6085-1, CLONTECH Laboratories)
was used. Unincubated, fertile, White Leghorn eggs (stage X according
to the classification by Eyal-Giladi and Kochav[10]) were used.
Blastoderms from 5 eggs were isolated from the yolk and washed several
times with Dulbecco's phosphate buffer (PBS, Dainippon Pharmaceutical
Co.) to remove as much yolk as possible. They were then dispersed by pipetting.
After dispersion, the cell clusters and debris were centrifuged at 400g
for 5 min at 4. The supernatant was discarded and cells were resuspended
in PBS mixed with 2.5 ng of pEGFP. After ten minutes, eletrotransfection
was done in the electroporation cuvette chamber (Gene Pluser Cuvette,
BIORAD). The mixture was ready to be exposed to the pulsant electric field.
Electroporation was performed using exponential electric pulses (field
strength 550 V, capacitance 25 F) generated by an electroporator (Gene
Pluse II, BIO RAD). Immediately after electroporation, the cell clusters
and debris were centrifuged at 400g for 5 min at 4 and the supernatant
was discarded. The precipitated cells were washed 3 times with Dulbecco's
Modified Eagle Medium (DMEM, GIBCO BRL) containing 10% bovine serum and
incubated at 37 for 12 hours. After the incubation, cells were suspended
in PBS.
Unincubated, fertile, Rhode Island red eggs (stage X embryos) were used. Blastoderms from 5 eggs were obtained by employing the same procedure as for loading cells. The treated blastodermal cells were suspended in PBS.
Electrofusion
was done in the two-phase polymer systems. The volume of the dextran phase
containing cells and loading materials was set at one-eighth of the total
volume and the rest was PEG phase. The two-phase solution was then vortexed
for a second to create an emulsion. Electroporation was performed using
three electric pulses (field strength 188 V/cm, half-time 1 sec) generated
by an electroporator (CUY21; TOKIWA Science, Japan). Immediately after
electroporation, the cell clusters and debris were centrifuged at 400g
for 5 min at 4 and the supernatant discarded. The precipitated cells
were washed 3 times with Dulbecco's Modified Eagle Medium (DMEM, GIBCO
BRL) containing 10% bovine serum.
Under
the fluorescent microscope, whether the fluorescent cells were fused with
non-fluorescent cells was confirmed. The fusion rate was determined by
the number of fused cells between fluorescent and non-fluorescent cells
in the total cell population, counted on randomly chosen microscopic fields.
2.2
Experiment 1
Unincubated,
fertile, White Leghorn and Rhode Island red eggs (stage X embryos) were
used. Blastoderms from 4 eggs in each strain were obtained. Washed and
dispersed Blastodermal cells were fused with electroporation in two-phase
polymer systems by employing the same procedure as described. The cells
precipitated by centrifugation were washed 3 times with DMEM containing
10% bovine serum. G1 glass capillaries
(Narishige) were pulled and beveled to a tip. Capillaries were filled
with 3-5 L of cell suspension (donor blastodermal cells).
Unincubated,
fertile, White Leghorn and Rhode Island red eggs (stage X) were employed
after being kept at 4 for 24-48 hours. Eggs were swabbed with 70% alcohol
and a 10-mm diameter
window was made at the sharp end just above the blastoderm. Fusing cell
suspension, 3-5 L, was injected by means of the capillary into the subgerminal
cavity of the recipient egg. After the injection, the window was closed
with adhesive tape and the eggs placed in an incubator (37, 50% relative
humidity, and turned through 90º/h) for 19 days. Then they were transferred
to a hatcher at 37 and 85% relative humidity until hatched.
2.3
Experiment 2
Unincubated,
fertile, white Leghorn or Rhode Island red eggs (stage X) were used. Blastoderms
from 3 eggs of one strain were obtained. The treated blastodermal cells
were centrifuged at the same condition as described above. The precipitated
cells were washed 3 times with DMEM containing 10% bovine serum. Donor
blastodermal cell suspension, 3-5 L, was aspirated by G1 glass capillaries.
Unincubated, fertile, different strain donor eggs (stage X) were used.
The procedures for the preparation of recipient eggs, the transplantation,
and the incubation methods were the same as described above.
2.4
Analysis of chimeric chicken
3 Results
Electrofusion
could be confirmed by fluorescent microscopy, since cells introduced a
marker gene GFP prior to fusion. Viability of blastodermal cells after
fusing with electroporation was approximately 50%. As for cell viability,
no significant differences were found between embryos electrically treated
and non-treated. The hatching rates of embryos were 3.4% for electroporation
and 1.4% for non-electroporation (Table 1).
Table
1. Embryonic viability and hatchability.
|
|
Viability |
||||
| d3 |
d7 |
d14 |
d21 |
Hatched |
|
| Trial
1 |
161/177
(91%) |
71/177
(40.1%) |
31/177
(17.5%) |
11/177
(6.2%) |
6/177
(3.4%) |
| Trial
2 |
67/70
(95%) |
29/70
(41.4%) |
6/70
(8.6%) |
4/70
(5.7%) |
1/70
(1.4%) |
| Control |
14/14
(100%) |
2/14
(14.3%) |
1/14
(7.1%) |
0/14
(0%) |
0/14
(0%) |
Trial
1: Eggs with electrofused blastodermal cells
Trial 2: Eggs with untreated blastodermal cells.
Control: Windowed eggs.
The
presence of color feather indicated that donor cells had contributed the
melanocyte pigmentation, which was observed in 1 bird out of 3 hatched
chicks. The bird grew up enough old to confirm pigmentation (Figure
1). In the embryos treated with electroporation, 2 embryos with testis
(Figure 2) died just before hatching.
These two birds were then subjected to determine W-chromosome-specific
DNA by PCR analysis. In one embryo, W-chromosome-specific DNA was detected
at the stomach, and
in the other, at the right and left gonads, large intestine, liver and
certain muscle, but not the stomach (Figure
3).
Figure
1. Chimeric chicken having donor-derived pigmentation. Left: 31-day
old chimeric chicken.
Right: 33-day old normal chicken.
Figure 2. Chicken embryos (A
& B) with testes.
Figure 3. PCR analysis of W-chromosome-specific
DNA from the tissues of embryos in Figure 2. (A) A 315-bp fragment specific
of W-chromosome-specific DNA detected in the genome of stomach (Lane
5) but not in the right and left side gonads (Lanes, 1 and 2), large intestine
(Lane, 3), liver (Lane 4) and muscle of each limb (Lanes 6-9).(B) A 315-bp
fragment specific of W-chromosome-specific DNA detected in right and left
gonads (Lanes 1 and 2), large intestine (Lane 3), liver (Lane 4), and
muscle of each limb (Lanes 6, 7, 8 and 9).Control is the blood from female
chicken (Lane f), and male chicken (Lane m).
4 Discussion
In this experiment, high percentage of mortality was consistently observed in the electrofused embryos. This might not be the consequence of injection or electroporation and may be due mainly to the opening of windows at the eggshell[6]. The same phenomenon was found in the embryos which were treated with non-electrical method. It is necessary to employ some high standard techniques for the production of chimeric chickens. In the present studies, donor blastdermal cells were distributed near the site of injection at the epiblast and subgerminal cavity. It is therefore recommended that cell injection should be as shallow as possible to increase the proportion of chimeric embryos. Some methods are needed to prevent blastodermal cells from escaping from the hole produced by injection[5]. Moreover, the developmental stage of recipient eggs or the sight for placing donor cells might have made some difference to the distribution of donor blastodermal cells. In this study, only one chimeric chick produced by electroporation indicated the real transfer of donor blastodermal cells. However, the existence of cells derived from donor eggs was confirmed by PCR analysis in the embryos whose gonads being testes. This finding suggested the possibility of producing somatic and germline chimeric chickens by transferring blastodermal cells fused with electroporation method. This result may also be caused by much more easy distribution of donor cells, because donor cells possessed the same membrane characteristics as recipient eggs and/or donor cell membrane might be broken temporarily to form minute pores. The efficiency of production of chimeric chicken was not discussed in the present experiment, since no embryos with non-electric fusion grew old enough to confirm the feather pigmentation. The PCR method could not be used for embryos with ovary to confirm chimera.
In
the present experiments, only one somatic chimera and two germline chimeras were
obtained when examined after hatching and/or some of the dead chicks just
before the hatching days.
Acknowledgements
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Correspondence
to: Dr.
Noboru Fujihara, Division of Animal Science, Faculty of Agriculture, Kyushu
University Graduate School, Hakozaki, Fukuoka 812-8581, Japan.
Tel/Fax: +81-92-642 2938
e-mail: nfujiha@agr.kyushu-u.ac.jp
Received
2000-06-16 Accepted 2000-10-30
