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Introduction
of DT40 cells into chick embryos
Mariko
Toba, Fumio Ebara, Hiroki Furuta, Yuichi Matsushimal, Yasuo Kitagawa1,
Noboru Fujihara
Animal
Resource Science Section, Division of Bioresource and Bioenvironmental
Sciences, Graduate School Kyushu University, Fukuoka, 812-8581 Japan
1Graduate Program for Regulation of Biological Signals, Graduate
School of
Bioagricultural
Science, Nagoya University, Nagoya,
464-8601 Japan
Asian J Androl 2001 Mar; 3: 49-53
Keywords:
DT40
cells; chick embryo; lacZ; polymerase chain reaction
Abstract
Aim: To examine the transfection of exogenous genes into chick embryos, applying the characteristics of avian leukosis virus (ALV)-induced chicken B cell line DT40 to the production of chimeric birds. Methods: The DT40 cells incorporated with exogenous gene (lacZ constructs encoding Escherichia coli -galactosidase: -gal) were introduced into chick embryos by the injection of cells into stage X blastoderm. Manipulated eggs were incubated for 3 (trial 1) or 6 (trial 2) days, and the expression of lacZ DNA was detected by a histochemical staining method of -galactosidase and polymerase chain reaction (PCR) analysis. Results: The survival rates of the manipulated embryos incubated for 3 days (stage 18-20: trial 1) and 6 days (stage 28, 30: trial 2) were about 42% and 38%, respectively.The expression rates of the lacZ gene in the embryos in the trials 1 and 2 were about 60% and 23%, respectively, for the survived embryos. Conclusion: The rate of embryonic viability and expression rate of introduced genes were not so high, but it suggested the possibility of utilizing the DT40 cells as a vector for carrying exogenous genes into chick embryos.
1 Introduction
During
the past several years, the production of transgenic animals has been
a prominent theme in animal biology. Transgenic mice have been shown to
be useful in the study of regulation and progress of mammalian development.
At the same time,
transgenic chickens have also been proved to be the excellent system for
studying developmental biology and could
be of great benefit to the poultry breeding industry. Transgenic mice
are produced routinely
by the method of microinjection of DNA into the pronuclei of the
fertilized ovum. However, the production of transgenic chickens may present a
set of problems that such experimental manipulation is impractical in
birds because of the unique reproductive anatomy and physiology of this
species. Therefore, a number of attempts have been conducted to create
transgenic birds by the use of a retrovirus as a vector[1].
This method, however, requires the handling of hazardous retroviruses
and limits the size of insert DNA. Other methods are lipofection[2]
and electroporation[3] for the producing of chimeras[4-6].
In these methods, however, introduced exogenous genes remained in episome
in the cells and persisted only transiently, suggesting that genes have
not been targeted. Therefore, it is necessary to create transgenic chickens
by homologous recombination and gene targeting methods. To circumvent
these serious problems, an alternative strategy should be developed for
producing transgened birds. One of these strategies is to use the DT40
chicken B cell line. The DT40 cells are avian leukosis virus (ALV)-induced
cell line, showing high level of homologous recombination and
rapid cell growth rate. Most of the gene integrations may occur at some
position on the chromosome at random (random integration), while chromosomal
DNA fragment in plasmids rarely undergo homologous recombination to the
DNA on chromosome containing homologous sequence, and as a result plasmid
DNA may be integrated into chromosome (targeted integration). The mouse
ES cells have been reported to produce target integration much more easily,
showing that random integration occur more 100fold frequency than the
case of targeted integration.
2 Materials and methods
2.1
Cell culture and transfection
The DT40 cells were maintained in Dulbecco's modified eagle medium (DMEM; Gibco, UK),supplemented with 10% fetal bovine serum (Gibco, UK) at 37 and 5% CO2 in the air. The cells were routinely split to 1106 per mL every other day. The LacZ-containing plasmids encoding Escherichia coli -galactosidase (-gal) were used as marker gene in this study. For transfection, 1107 cells were suspended in 500 mL PBS containing 25 mg linearized plasmid and electroporated with a Gene Pulser apparatus (Bio Rad) at 550 V and 25 F. Following electroporation, the cells were transferred to 20 mL fresh medium lacking antibiotics and incubated for 24 hr. The cells were then resuspended in 80 mL medium containing a final concentration of 1.5 mg/mL G-418 (Geneticin) and divided into 96-well plates. After 7-10 days, drug-resistant colonies were obtained (Figure 1).
Figure
1. Micrographs of DT40 cells. A: normal; B: assessment of exogenous
-gal
activity in DT40 cells. Bar=10 m.
2.2
Injection into embryos
Unincubated
and fertilized White Leghorn eggs (stage X)[11] were
used throughout the experiments. The eggs were operated as follows: a
window of about 10 mm in
diameter was opened at the sharp edge of eggshell and certain amount of
thick albumen around the germinal disc was removed by aspiration. Approximately
300-500 DT40 cells in 1-2 mL medium were injected into the subgerminal
cavity using a fine glass micropipette. The windows were then sealed with
a Scoctch tape. Manipulated eggs were incubated for 3 or 6 days while
rocking intermittently at anangle of 90 degree in hourly cycles at 37-38,
under a relative humidity of 60-70%. As controls, several fertilized eggs
were incubated without injection. The embryos
were cultured to reach stage 18-20 (trial 1) and stage 28-30[12](trail 2).
After a given period of incubation, the embryos were fixed with PBS, rinsed,
and stained for -galactosidase activity as described below.
2.3
Assessment of -gal activity
The
expression rate of lacZ DNA was detected by a histochemical staining method
of -galactosidase. Embryos and extra-embryonic tissues at stage 18-20
and 28-30 were removed from the yolk, washed and immersed in PBS and fixed
for 20 min in PBS,
pH 7.4, containing 0.25% (v/v) glutaraldehyde at room temperature. Fixed
embryos and extra-embryonic tissues were washed several times with PBS,
incubated at 37
for about 1 hr in a solution containing 20 mg/mL 5-bromo-4 chloro-3 indolyl--D-galactopyranoside
(X-gal, Sigma-Aldrich, Tokyo), 50 mmol/L potassium ferricyanide, 50 mmol/L
potassium ferrocyanide, 1 mol/L MgCl2, and 10% (v/v) triton
X-100 in PBS.
2.4
PCR analysis
3 Results
The survival rates of the manipulated embryos incubated for 3 days (stage 18-20: trial 1) and 6 days (stage 28-30: trial 2) were ca. 41% and 38%, respectively. The expression of the lacZ gene in the embryos from trial 1 (Figure 2a), as revealed by -galactosidase activity, was ca. 60% for the survived embryos. In trial 1, most of the expression of DNA was detected in the extra-embryonic tissues surrounding the embryos (ca. 86%), but the intra-embryonic expression of lacZ gene was ca. 18%. The intra-embryonic expression sites of introduced DNA were mostly in the head and breast of the embryos. The level of gene expression in the embryos in trial 2 was ca. 23% of the survived embryos, much lower than that in trial 1. In the trial 2, the extra-embryonic expression of lacZ gene was ca. 60%, and the intra-embryonic expression (Figure 2b) was ca. 40%. The main expression site of DNA in the embryos was only the breast for the present experiments (Table 1). When the PCR analysis was applied, some of the treated embryos showed positive lacZ activity. (Figure 3).Figure
2. X-gal stained chick embryos: A Trial 1 (1): stage 18-20, arrowheads
indicate X-gal-stained spots; B Trial 2 (5): stage 28-30, arrowheads
indicate X-gal-stained spots; C (1); D (9).
Figure 3. Amplified products spearated
through an agarose gel after polymerase chain
reaction (PCR) of lacZ DNA. Lane M: marker; Lane P: positive control;
Lane N: negative control; Lanes 1 to 11: sample DNA.
Table
1. Detection of exogenous gene (lacZ) in the embryos injected with DT40
cells (stained with X-gal sdution).
|
|
No.
of survived |
DNA
expression |
Sites
of gene expression |
||
|
Head |
Heart |
Extra-embryonic |
|||
|
Trial
1 |
37/90
(41.1) |
22/37
(59.5 |
2 |
2 |
19 |
|
Trial
2 |
22/58
(37.9) |
5/22
(22.7) |
0 |
2 |
3 |
4 Discussion
The DT40 cell line has been shown previously to undergo homologous recombination at an exceptionally high frequency[7]. Therefore, this characteristics of the cells were examined for the transfection of exogenous genes into chick embryos. No studies have so far been reported about the introduction of DT40 cells into the living animals. At first, it was investigated whether DT40 cells may take root into the chick embryos by using the method for producing chick chimeras. In this method, chicken blastodermal cells carrying exogenous genes were injected into the subgerminal cavity of stage X chick embryos in a manner that has been shown to produce somatic and germline chimeras[2,4,14,15]. Then this method was employed for the introduction of exogenous cells into the chick embryos, and subsequently the same method was applied to the DT40 cells. In the results of this experiment, the viability of both trial 1 and 2 embryos was approximately 40%, with the occurrence of some deformities. Expression rates of injected lacZ gene in trials 1 and 2 were about 60% and 23%, respectively, and the expression sites of DNA in extra-embryonic tissues were wider than those of embryonic tissues. In the previous reports, exogenous marker gene (the -actin-lacZ/MiwZ) in the transfection reagent was injected into the blastodisc of unincubated fertilized eggs[16], showing a similar result with this experiment. According to the reports on the distribution of blastodermal cells transferred to chick embryos for chimera production, much more donor blastodermal cells were located in the yolk than in the subgerminal cavity, and the cells were also found outside the epiblast because they had escaped from the small hole left by pipetting[17]. The facts from this results that expression sites of injected DNA in the extra-embryonic tissues were wider than those of embryonic tissues suggest that the DT40 cells injected into subgerminal cavity might have escaped from the pinhole and were mixed with extra-embryonic tissues during the embryonic development, though the mechanism for this phenomenon is not clearly explained yet. The DNA expression rate of trial 2 had been significantly (P<0.05) lower than that of trial 1. Another report, showing that the transfer of primordial germ cells (PGCs) containing transfected exogenous gene to the recipient embryos has been shown that the DNA expression rates were reduced during the embryonic development[18]. One of the reasons was that the embryos were stained by X-gal with extra-embryonic tissues containing vitelline artery in the trial 1, while embryos in the trial 2 grew up larger than those in trial 1, being less stained in only a few extra-embryonic tissues. However, the fact that DNA expression rates were confined to a small scale, indicating the decreased expressions of genes in the embryonic tissues, demonstrated that the injected DT40 cells might be excluded from the embryos due in part to immunological surveillance.
Therefore, in the present experiments, the viability of treated embryos and the expression rates of introduced DNA were not successful. However, this result suggests the possibility of introducing the DT40 cells into chick embryos to create a kind of chimericchicken in the future. It may be concluded that the DT40 cells were worth utilizing their features as a vector carrying exogenous genes into chick embryos. The physiological effects of the DT40 cells on chick embryos remain to be elucidated.
Acknowledgements
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Correspondence
to: Dr.
Noboru Fujihara, Animal Resource Science Section, Division of Bioresource
and Bioenvironmental Science, Graduate School Kyushu University, Fukuoka
812-8581 Japan.
Tel/Fax: 81-92-642 2938
e-mail: nfujiha@agr.kyushu-u.ac.jp
Received
2000-08-07 Accepted 2000-12-22
