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Reproductive
characteristics of transgenic (TG) chickens carrying an exogenous gene
Fumio
Ebara, Noboru Fujihara
Animal
Resource Science Section, Division of Bioresoure and Bioenvironmental
Sciences, Graduate School of Kyushu University, Fukuoka
812-8581, Japan
Asian J Androl 1999 Sep; 1: 139-144
Keywords:
exogenous
genes; chicken; MiwZ; germinal crescent; primordial
germ
cells; spermatozoa; transgenic animals
Abstract
An exogenous gene (lacZ/MiwZ) introduced into the germinal crescent region (GCR) of avian embryos was confirmed to be successfully transferred to the gonads via the primordial germ cells (PGCs). Following hatching, the chicks were raised until the stage of sexual maturation. The incorporation of MiwZ DNA was detected in male and female transgenic chickens, respectively. The normal male and female transgenic birds were subjected to artificial insemination according to routine methods. Fertilized eggs obtained from female transgenic chickens were incubated for 72 h and the embryos removed from the yolk were examined by X-gal staining to detect the introduction of MiwZ in the offspring. As a result, the expression of MiwZ was detected in the offspring. Furthermore, the presence of MiwZ in the extracts from embryos was also detected by polymerase chain reaction (PCR) analysis. In male transgenic chickens, the presence of injected MiwZ in the extracts from sperm was also confirmed. The exogenous gene introduced into the GCR migrated successfully to the gonad resulting in its incorporation into the offspring and spermatozoa of transgenic chickens.1 Introduction
In
the nineteen-eighties, since transgenic chickens have been produced successfully
using retroviruses as a vector[1-4], various trials have been
done to produce transgenic chickens[5-8]. However, the retrovirus
method has disadvantages such as limitation in size of the inserted gene
and the method has been considered to be dangerous. Thus, non-retroviral
methods, such as direct
DNA transfer into chicken embryos have been succeeded by microinjection
of the gene into the germinal disc of the fertilized ovum[9,10],
microinjection into the blastoderm (stage X)[11] in ovo[12]
and the transfection of blastodermal cells[13,14].
Because
the production of germline chimera was a powerful method to produce transgenic
chickens, germ cells were useful tools for this purpose. The germ cells
are located far from the gonad at the
early developmental stage, existing as primordial germ cells (PGCs).
In
avian species, the PGCs circulate temporarily in the bloodstream before
migrating into the germinal ridge[15-18]. Thus, the technique
for collecting PGCs from blood vessels has been established[19].
Since the PGCs are progenitor cells of ova or spermatozoa, an attempt
has been made to introduce exogenous genes into chicken embryos via PGCs[20,21].
2 Materials and methods
2.1
Preparation of fertilized eggs
Fertilized
eggs of Rhode Island Red Chickens were obtained by artificial insemination
and collected daily. Fertility was more than 80% in this study.
2.2
Preparation of the DNA solution
Figure
1 showed the structure of the constructed plasmid MiwZ maker gene,
which contains the E. coli.--galactosidase gene
(lacZ) under the control of RSV enhancer and the chicken -actin gene
promoter/enhancer[24]. MiwZ DNA (5 L) was
mixed with 22.5 L of transfection reagent (DOTAP/l781995; Boehringer
Mannheim, Germany), and then diluted up to 50 L with Hepes-buffered
saline (HBS; 20 mmol/L Hepes containing l50 mmol/L NaCl, pH7.4) in a polystyrene
tube/ The DNA mixture was incubated for 10-15 min at room temperature
(22-25). A micropipette (G-1, Narishige, Tokyo, Japan), whose tip had
an outside diameter of about 40 m, was filled with the DNA solution
prior to microinjection.
Figure 1. Structure of the recombinant plasmid, MiwZ. -actin (-act) and -crystallin (-cry) sequences are shown by the wavy line and their exon sequences by the solid boxes. Bacterial sequences (-gal) are indicated by the open box, HSV-tk sequences by the vertically striped box, SV40 sequence by the horizontally striped box, the RSV LTR sequences by the open arrow and plasmid vector sequence by the solid horizontal line. Promoters and terminaiton signals (tk ter and SV ter) are also indicated. the restriction sites are indicated by B, BamHI; E, EcoRI; K, KpnI; Ps, PstI; Sc, ScaI; SI, Sall; Sm, SmaI; Sp, SphI; and Xb, XbaI.
2.3
Microinjection of the DNA solution
Fertilized
eggs were incubated at 38.5 under a relative humidity at 60%-70%. A
window of 10-15 mm diameter was opened at the shape end of egg shell when
embryos reached the stage 3-5 (trial 1) or 9-11 (trial 2) of development.
For trial 1, DNA solution was injected into the site near the GCR with
an approximately 1.0 L in total volume, and for trial 2, the solution
was introduced into the GCR with the same amount of DNA. After the injection,
the window was closed by adhesive tape and the incubation was continued
until hatching. Hatched chicks were raised until the stage of sexual maturation.
2.4
Detection of lacZ gene expression in offspring
The
expression of DNA (lacZ/MiwZ) was detected by a histochemical staining
method (X-gal staining). Fertilized eggs obtained from female transgenic
chickens were incubated at 37.8 under a relative humidity of 60%-70%
for 72 h (stage 19-20). The embryos were removed from the yolk and washed
with DPBS(-)(DULBECCOS PBS TABLETS without Ca2+ and Mg2+,
8l726-001; Dainippon Pharmaceutical Co., Laboratory Products Division).
The embryos were fixed with 0.25 % glutaraldehyde in DPBS(-) for 20 min
at 4 and then washed three times with DPBS(-). The embryos were stained
for 3-6 h or over night at room temperature (22-25) with a staining
solution containing 5 mmol/L potassium ferricyanide, 5 mmol/L potassium
ferricyanide, 0.2 mmol/L MgCl2, 0.1% TritonX-100 and 0.1% 5-bromo-4-chloro-3-indolyl--D-galactopyranoside
(X-gal, Sigma, USA) in DPBS(-) (pH7.8).
2.5
Detection of lacZ gene by PCR analysis
3 Results
3.1
Fertility of offspring
In
the fertilized eggs obtained from four female transgenic chickens, fertility
of only one chicken (transgenic chicken 18) was lower 20%-30% than that
of the others (Table 1).
Table
1. Fertility of eggs obtained from the chimeric chickens.
|
Chimeric
chicken |
Number
of embryos manipulated |
Fertility
(%) |
|
395 |
36 |
34/36
(94.4) |
|
398 |
47 |
44/47
(93.6) |
|
18 |
48 |
30/48
(62.5) |
|
17 |
49 |
42/49
(85.7) |
3.2
Expression of lacZ gene in offspring
Expression of lacZ gene was detected in the embryos of offspring (Figure 2). In this study, the expression of DNA was detected in the offspring of all transgenic chickens (Table 2). The expression of lacZ gene decreased gradually as the time proceeded in this experiment. Finally, no expression was detected in the late embryos.
Figure
2. Expression sites of MiwZ DNA in embryos from offsprings by X-gal
staining (arrows). (A) and (B): stage 19-20. Bar=250 m.
Table
2. Expression of MiwZ-DNA in the embryos from offspring determined by
X-gal staining method.
|
Chimeric
chicken |
Number
of embryos manipulated |
Rate
of MiwZ-DNAexpression |
|
395 |
34 |
4/34
(11.8) |
|
398 |
40 |
2/40
(5.0) |
|
18 |
30 |
3/30
(10.0) |
|
17 |
42 |
2/42
(4.8) |
3.3
Presence of lacZ gene by PCR analysis
In
female transgenic chickens, the lacZ gene was detected in the embryos
of offspring (Table
3, Figure 3).
Table
3. Detection of MiwZ-DNA in the embryos from offspring by PCR analysis.
|
Chimeric
chicken |
Number
of embryos manipulated |
Present
rate of lacZ band/% |
|
395 |
34 |
7/34
(20.6) |
|
398 |
44 |
7/44
(15.9) |
|
18 |
30 |
5/30
(16.7) |
|
17 |
42 |
7/42
(16.7) |
Figure 3. Detection of lacZ gene in embryos from offsprings by PCR analysis. M: marker, P: positive control, N: negative control, 1-7: samples.
In
this experiment, the DNA band was also detected in the extracts from the
embryos which did not express by X-gal staining. In male transgenic chickens,
the lacZ gene was
also detected in sperm cells by PCR analysis (Table 4,
Figure 4).
Table
4. Detection of MiwZ-DNA in spermatozoa from chimeric chickens by PCR
analysis
|
Chimeric
chicken |
Number
of spermatozoa/mL |
Expression
rate/% |
|
15 |
4.2109 |
3/6
(50) |
|
394 |
11109 |
1/7
(14.3) |
Figure
4. Detection of lacZ gene in spermatozoa from male chimeric chickens
by PCR analysis. M: marker, P: positive control, N: negative control,
1-10: samples.
4 Discussion
Transgenic
chickens obtained from exogenously DNA-injected embryos showed a normal
appearance, giving a regular laying performance, although the rate of
egg production was low. In the case of male chickens, semen production
also normal, appeared to be
showing a natural appearance of semen characteristics (data not
shown).
In
the present study, the expression of exogenous gene (lacZ/MiwZ) was detected
by the methods of X-gal staining in the embryos obtained from the offspring,
although the most remarkably detected DNA was by the method of PCR analysis.
Judging from these findings, it was clearly shown that exogenous DNA injected
into avian GCR was successfully introduced into the gonads and remained
until sexual maturation. The introduced DNA was obviously incorporated
into the offspring of transgenic chickens, resulting in the detection
of the gene in spermatozoa.
In
the present experiments, percentages of offspring showing a positive reaction
of lacZ gene were approximately 10% for the first two months and no positive
reaction for the chickens after four months. In this study, as mentioned
above, the DNA expression was decreased as the time of the experiments
elapsed. It has been reported that the expression of exogenous DNA in
chicken embryos was gradually decreased and was not transmitted to the
next generation[21]. In the present results, the same tendency
was observed, through introduced exogenous DNA being clearly expressed
in the offspring (Figure 2, Table
2).
On
the one hand, some of the embryos having no positive reaction to X-gal
staining showed the presence of introduced DNA by the method of PCR analysis,
suggesting that in some of the successfully transmitted offspring, the
expression of lacZ gene might be suppressed through generative transmission.
With
regard to transgenic male chickens, spermatozoa obtained from the offspring
showed the presence of exogenous gene. It is not clear whether or not
the spermatozoa might retain physiological function when used for artificial
insemination. This uncertainty arises because the introduced gene might
not be integrated into genome DNA. On the other hand, an advantage of
our method for introducing foreign genes into chicken embryos was that
no sacrifice of laying hens was needed to obtain the ova from the oviducts.
In the previous methods, fertilized ova (single-cell stage) were obtained
from the magnum of the oviducts by a surgical operation[9,10].
On
the contrary, a possibility has been reported to introduce an exogenous
gene into the embryonic tissue using incubated fertilized eggs without
sacrificing hens[12]. Another useful method was that the injection
of foreign DNA into GCR was also successful for transferring a foreign
gene into the gonads[22].
The
transferred gene was detected in gonadal tissue from one and two-month-old
chickens[23]. The results from this study showed that the chicken carrying
transferred foreign gene was successfully transmitted to the offspring
and spermatozoa from transgenic chickens.
Since
PGCs are progenitor cells of ova or spermatozoa and ensure the transmission
to the next generation, many trials, have been done to develop some techniques
for gene transfer to chicken embryos via PGCs[15,25,21]. However,
most of the previous reports showed that expression of the introduced
DNA has not been detected in
the next generation of chickens. The present method described here, therefore,
suggests the possibility of the successful production of transgenic chickens
in the future.
5
Acknowledgments
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Correspondence
to Noboru Fujihara, Ph.D.
Tel & Fax: +81-92-642 2938
E-mail: nfujiha@agr.kyushu-u.ac.jp
Received
1999-05-18 Accepted 1999-08-22
