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NAITO Blackwell Science, LtdOxford, UKASJAnimal Science Journal1344-39412003 Blackwell Publishing Asia Pty LtdJune 2003743157168Review Article
REVIEW ARTICLE Development of avian embryo manipulation techniques and their application to germ cell manipulation
National Institute of Agrobiological Sciences, Tsukuba-shi, Japan
The development of chicken embryo culture techniques, from single-cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single-cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non-viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efﬁcient system for producing transgenic chickens by combining it with the highly efﬁcient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary.
KEYWORDS: chicken, embryo manipulation, germ cell, germline chimera, transgenesis.
The avian embryo provides an excellent model for studying embryology, especially in the amniotes of higher vertebrates. Manipulation of early avian embryos has been hampered, however, by their distinctive reproductive characteristics. The chicken ovum is fragile and contains a large amount of yolk. Fertilization takes place within 15 min of ovulation and early embryonic development proceeds in the hen’s oviduct during the egg formation process. After oviposition, embryonic development proceeds inside the eggshell. Development of a chicken embryo culture technique from the single-cell stage to hatching (Perry 1988) enables the manipulation of early stage embryos which produce viable offspring. Using this embryo culture technique, various attempts have been made to produce transgenic chickens as well as germline chimeric chickens. Genetic manipulation of chickens provides great potential for the basic sciences as well as practical
applications. Production of pharmaceutical materials in the eggs of transgenic chickens is the ﬁrst target application, and success in this production system could lead to the commencement of a new industry. Manipulation of early chicken embryos has been reviewed elsewhere by Naito (1997, 1998, 2003), and manipulation of quail embryos has been reviewed by Ono (1997, 2001). In this review, recent studies on chicken embryo culture and embryo manipulation techniques for manipulation of germ cells are described. The developmental stages of chicken embryos from ﬁrst cleavage to primitive streak formation (stages I to XIV) are designated by roman numerals (Eyal-Giladi & Kochav 1976), and from prestreak stage to hatching
Correspondence: Mitsuru Naito, National Institute of Agrobiological Sciences, Tsukuba-shi, Ibaraki-ken 305–8602, Japan. (Email: email@example.com) Received 28 October 2002; accepted for publication 16 January 2003.
Animal Science Journal (2003) 74, 157–168
thick albumen (1. collected from the uterus of the oviduct. 1). resulting in a 22.036) and thin albumen (1. embryogenesis (phase II) for three days. II. In order to improve the hatch rate of cultured embryos. increasing the hatch rate to 34.M.029). During these periods. A fertilized ovum obtained from the posterior portion of the magnum of the oviduct at 2.5∞C for 24 h.4% by culturing embryos from the single-cell stage to hatching. Then the thick albumen and culture medium are System I 4-day incubated embryo System II 8-day incubated embryo System III 22-days at hatch Fig. A single-cell stage embryo is cultured in vitro using 3-step culture techniques (systems I.75 h after the preceding egg is laid is cultured in a glass vessel with a small amount of thin albumen (system I). Animal Science Journal (2003) 74. II and III). and obtained a 7% hatching rate by culturing embryos from the single-cell stage to hatching. the differences in the speciﬁc gravity among the yolk (1. 1994).5% hatch rate (Naito & Perry 1989). Naito et al. The embryo develops normally and hatches after 22 days in culture. EMBRYO CULTURE Chicken embryo development from fertilization to hatching can be divided into three phases: fertilization to blastoderm formation (phase I) for one day. and embryonic growth (phase III) for 18 days. At this stage the yolk is surrounded by a small amount of thick albumen. 157–168 158 . Perry (1988) devised three discrete culture systems (systems I. The modiﬁed method of chicken embryo culture from the single cell stage to hatching is as follows (Fig.040) play a very important role in the normal development of chicken embryos (Suda et al. and III) to meet the changing demands at successive stages of development. This technique can be applied to culture embryos during the early cleavage stages (stages I–II). NAITO (stages 1 to 46) by arabic numerals (Hamburger & Hamilton 1951). (1990) modiﬁed Perry’s culture system. 1 In vitro culture system for chicken embryos. The fertilized ovum is incubated at 41–41.
the future gonads (Nieuwkoop & Sutasurya 1979. Naito et al. the contents of the reconstituted egg are transferred to a larger recipient eggshell with air space. PRODUCTION OF GERMLINE CHIMERIC CHICKENS Development of the chicken germline From the analysis of germline-speciﬁc expression of the chicken Vasa gene. Kuwana 1993. The percentage of donor-derived offspring from chimeric chickens was. Using the chicken embryo culture technique increases accessibility of the embryos because the whole embryo can be exposed. 1993. 2/719) because recipient embryos possess their own PGC and the proportion of donor PGC to recipient PGC is low. however. The cultured embryo is then incubated using systems II and III. using a piece of gauze overlaying the whole egg (Naito et al. This procedure was used to produce somatic and germline chimeric chickens (Petitte et al. A fertilized ovum with a thin layer of thick albumen obtained from the anterior portion of the magnum of the oviduct 60–80 min after the preceding egg is laid can be cultured in system I. The egg is incubated at 37.5% hatch rate (Naito & Perry 1989. and sometimes the number of donor PGC dominates the recipient PGC. a reduction in the number of PGC in recipient embryos and an increase in the number of PGC in donor blastodermal cells is required. ﬁlled with thin albumen.8∞C and 50–60% relative humidity for 14 days with rocking. and then incubated in stationary position at 37. 1990). 1990). germ plasm (Tsunekawa et al. These Vasa-positive cells. called primordial germ cells (PGC). However. PGC migrate to the germinal ridges. secondary oocytes and ova (Naito 1998). secondary spermatocytes. Primordial germ cells which enter the testes differentiate into spermatogonia. eggshell membranes and eggshell are formed around the ovum and. Primordial germ cells translocate gradually to the dorsal side of the hypoblast at stages XI–XIV. To reduce the number of PGC in stage X recipient embryos.Manipulation of avian embryos removed and only the yolk is transferred to the recipient eggshell. and Vasa protein is restricted to the basal portion of the cleavage furrow during the early cleavage stages. and the opening of the eggshell is covered with cling ﬁlm (system III). The manipulated embryos were incubated until hatching. where they start to differentiate into male or female gametes. Thereafter. 1996). are located in the ventral surface of the area pellucida (Karagenc et al. When the blood vascular system develops. primary spermatocytes. very low (0. Use of g-ray irradiation delays development of the treated embryos by approximately 24 h. Development of the chicken embryo culture technique from the single-cell stage to hatching enables access to developing embryos at any developmental stage. spermatids and spermatozoa. embryo culture systems II and III are used to obtain a 50. At stage X. 1994). Kuwana & Roglska 1999). Blastodermal cell transfer Blastodermal cells at stage X contain PGC or their precursors as well as somatic cells with pluripotency.0–62. the chicken germline is thought to be determined by maternally inherited factors in the Animal Science Journal (2003) 74. 1997a. 2000). irradiation by g-ray proved effective in modifying the recipient embryos (Carsience et al. The Vasa-positive structure is predominantly localized in the oocytes. Finally. The hatch rate was reported to be 19. the fertilized ovum can be transferred into the oviduct of a recipient hen (Tanaka et al. When three-day incubated embryos (stage 18) are cultured.3%. and are carried anteriorly to the germinal crescent region. 157–168 159 . 1995). Thoraval et al. 1994). during which time the injected donor blastodermal cells containing PGC increase in number in the recipient embryo. and secured with plastic rings and elastic bands (system II).6∞C and 60–70% relative humidity for another four days until hatching. When embryos are cultured from the laid egg stage (stage X). The reconstituted egg is incubated at 38∞C and 50–60% relative humidity for three days with rocking. The PGC which enter the ovary differentiate into oogonia. approximately 30 Vasa-positive cells are scattered in the central disc of the area pellucida. Stage X blastodermal cells isolated from the yolk of freshly laid eggs were dissociated and transferred into the subgerminal cavity of a recipient blastoderm (Etches et al. Speksnijder & Ivarie 2000). PGC enter the blood vessels and circulate temporarily throughout the embryo. 1993. In order to increase donor-derived offspring from germline chimeric chickens. covered with cling ﬁlm. primary oocytes.5%. Naito & Kuwana 2003). Thick albumen. A technique for in vitro culture of an ovum just after fertilization without thick albumen. or in vitro fertilized ova has not yet been devised for chickens. embryo culture system III is used. Manipulation of chicken embryos from stage X onward can also be done by opening a small hole in the eggshell (Etches et al. the embryo develops normally inside the eggshell through to hatching. after laying.
These studies showed that most PGC originate from the central disc. 2). and area opaca were transferred into recipient blastoderms and the donor cell contribution to the recipient germline was assessed. 1998). The area pellucida is subdivided into a central disc and a peripheral marginal zone. 1997. (1994a) removed embryonic blood from the recipient embryos prior to donor PGC injection. Removing a cell cluster from the central disc of a stage X recipient blastoderm and then injecting donor blastodermal cells. (2001a) also analyzed the distribution of PGC in stage X blastoderms. and genetic factors may affect this variation (Tajima et al. Tajima et al. Ginsburg and Eyal-Giladi (1987. The efﬁciency of obtaining donor-derived offspring from these chimeric chickens was low. However. According to these results. 1999. however. 1997. Avian PGC have a unique migratory pathway and various attempts have been made to produce germline chimeric chickens by the transfer of PGC obtained from the germinal crescent region (Wentworth et al. Primordial germ cells were successfully transferred into recipient embryos and differentiated normally into functional gametes and gave rise to viable offspring. The results showed that most of the PGC arise from the central disc. 1989. application of busulfan (Aige-Gil & Simkiss 1991b. Naito et al. from embryonic blood (Simkiss et al. Vick et al. Hallet & Wentworth 1991. 1991a. A stage X blastoderm is composed of a single-layered area pellucida and a peripheral area opaca. and the female is the heterogametic sex (ZW). Naito et al. 2003). They isolated and cultured various fragments of blastoderms in vitro and then determined the presence of PGC by periodic acid–Schiff staining. including ultraviolet irradiation (Reynaud 1976. which appeared to increase the efﬁciency of obtaining donor-derived offspring from the chimeric chickens. (2001a) failed to replace the recipient PGC with donor PGC efﬁciently with this technique in chimeric chickens. the average frequency of donor-derived offspring was 81% for three male chimeric chickens and 96% for one female chimeric chicken. 1993a). marginal zone. Naito et al. additional experiments are needed to reconcile these observations. Furthermore. The dissociated blastodermal cells derived from the central disc. the male is the homogametic sex (ZZ). producing spermatozoa with the Z chromosome. 2000). the distribution of PGC in stage X blastoderms needs to be analyzed. Zhao et al. 1996. and approximately 3. but cells from the marginal zone and area opaca also contain PGC although the number is very small. Naito et al. 1989) and Ginsburg (1994. 157–168 160 .M. These chimeric chickens continued to produce donor-derived offspring for a maximum of 146 weeks without any sign of immune rejection (Naito et al. (1994a) succeeded in producing germline chimeric chickens with a high transmission rate of donor-derived gametes by transferring PGC into partially sterilized recipient embryos (Fig. Animal Science Journal (2003) 74. especially blastodermal cells derived from the central disc. The number of PGC in the bloodstream of developing embryos shows great variation. 2001a).5 times that for transfer from BPR to WL (23% for six male chimeric chickens). All these methods also affect embryonic development and donor PGC. Vick et al. application of concanavalin A (Al-Thani & Simkiss 1991). and excision of the germinal crescent region (McCarry & Abbott 1982). it is necessary to increase the number of donor PGC and sterilize the recipient embryos. Mixed-sex germline chimeric chickens In avian species. 1992) by which germline chimeric chickens were produced at an efﬁciency of donor-derived offspring of up to 12% (Tajima et al. twin embryos were occasionally produced by this method and the occurrence of twin embryos reduces the number of embryos available for chimera production as a result of high mortality and failure to hatch (Naito et al. Primordial germ cell transfer Germline chimeric chickens can be produced efﬁciently by manipulating PGC directly (Naito & Kuwana 2003). NAITO To increase the number of PGC in donor blastodermal cells. 1993). because of an insufﬁcient number of PGC transferred to the recipient embryos. laser irradiation (Mims & McKinnel 1971). Several methods have been used to reduce or eliminate endogenous PGC from recipient embryos. A method of concentrating PGC from embryonic blood has been devised (Yasuda et al. 1997) analyzed the origin of PGC in stage X blastoderms. successfully replaced the recipient PGC with donor PGC (Kagami et al. 1995. 1999. When PGC were transferred from White Leghorn (WL) embryos to Barred Plymouth Rock (BPR) embryos. Although this technique seems to be effective for producing germline chimeric chickens. AigeGil & Simkiss 1991a). To further increase the efﬁciency of germline transmission of donor PGC in germline chimeric chickens. 1989) or from embryonic gonads (Chang et al. 1993b). 1998a). donor blastodermal cells should be collected from the central disc of stage X blastoderms for producing germline chimeric chickens.
1995. Naito et al. Simkiss et al. Both same-sex and mixed-sex germline chimeric chickens produced donor-derived offspring efﬁciently. The transferred PGC-derived offspring can be produced by mating the germline chimeric chickens. Using PCR. or PGC obtained from embryonic blood.5-day incubated embryos) into the same and opposite-sex recipient embryos. but few donorderived offspring were obtained from mixed-sex germline chimeric chickens. PGC isolated from embryonic blood are transferred into recipient embryos and produced germline chimeric chickens. the transferred PGC differentiated normally into functional gametes. and clariﬁcation of this issue is required. But when the sex of the donor PGC was different from the recipient embryos. (Kagami et al. The mixed-sex germline chimeric chickens provide an excellent system for studying molecular mechanisms of sexual differentiation and germ cell differentiation (Stevens 1997). amplifying the W chromosome-speciﬁc repeating sequences (Mizuno et al. Naito et al. 1996. Same-sex and mixed-sex germline chimeric chickens have been produced by the transfer of stage X blastodermal cells (Kagami et al. the differentiation of donor PGC into functional gametes appeared to be restricted to a greater degree. However. indicating that PGC at stage X have the ability to differentiate into both male and female gametes. 1997). (1999) transferred PGC obtained from embryonic blood (2. 1997a. Naito 1998).Manipulation of avian embryos Germline chimeric chickens Transfer PGCs Mating Stages 13–15 embryo Stages 14–15 embryos Donor-derived offspring Fig. 1995. Chicken embryos can be sexed using PCR (Clinton 1994. The stage X blastodermal cells contain PGC or their precursors. 2001). the differentiation of PGC in mixed-sex germline chimeric chickens still remains unclear. The combination of WL donor and BPR recipient produced W-bearing Animal Science Journal (2003) 74. Furthermore. Tagami et al. producing ova with either the Z or W chromosome. When the sex of donor PGC was the same as the recipient embryos. Clinton et al. at some developmental stages. giving rise to viable offspring via germline chimeric chickens. into male recipient embryos. 2 Production of germline chimeric chickens by primordial germ cell (PGC) transfer. Southern hybridization. Naito et al. Thus. Recent development of chicken embryo manipulation techniques has made it possible to produce mixed-sex germline chimeric chickens by the transfer of stage X blastodermal cells or PGC (Etches et al. male or female donor blastodermal cells were transferred into the same or opposite-sex recipient embryos and same-sex or mixed-sex germline chimeric chickens were produced. 1993). 157–168 161 . In these experiments. W chromosome-speciﬁc repeating sequences were occasionally seen in DNA extracted from the semen of male chimeric chickens produced by transfer of stage X female blastodermal cells. irrespective of their genetic sex. (2001a) carried out similar experiments and found that PGC at stage X differentiated normally in samesex germline chimeric chickens. 1999). 1997. and in situ hybridization.
W-bearing spermatozoa were also identiﬁed in sex-reversed chickens (genetically female and phenotypically male) produced by injecting aromatase inhibitor into the eggs on day 5 of incubation (Abinawanto et al. If these W-bearing spermatozoa have the ability to fertilize ova. manipulation of gonads in vivo. and the hatchlings contained two populations of spermatogonia derived from the transferred quail PGC and endogenous chicken PGC (Ono et al. 2001). the sex ratio of offspring could be altered. Perry et al. is efﬁciently expressed in the developing embryos. The fertilizing ability of these W-bearing spermatozoa is currently unknown. non-viral methods for DNA transfer into chickens are preferable. but it persists episomally and is gradually lost during embryonic development. 2002). 1998). Quail–chicken interspeciﬁc chimeras have been produced by the transfer of stage X blastodermal cells (Naito et al. 1992). The injected DNA. but is not directly injected into the male or female pronucleus because of the opaque cytoplasm. and the injected DNA is gradually lost during embryonic development (Sang & Perry 1989. NAITO spermatozoa more efﬁciently than the reverse combination (Kagami et al. 1991. maturation periods and immunological acceptance need to be considered. Naito 1997. 1998a). such as by microinjection of DNA into fertilized ovum. Sang 1994). Quail–chicken interspeciﬁc chimeras have also been produced by the transfer of PGC isolated from embryonic blood (Yasuda et al. Introduction of exogenous DNA into chickens can be carried out. injected DNA has been expressed strongly in developing embryos and has occasionally been detected in blood cells or spermatozoa of adult chickens (Naito et al. incubation periods. For practical applications. and many transgenic chickens have been produced by retroviral vector methods (Ronfort et al. Microinjection of DNA into fertilized ova Manipulation of a fertilized ovum at the single-cell stage has become possible by devising chicken embryo culture techniques from single-cell stage to hatching (Perry 1988. Stages 14–15 Manipulation of PGCs Chicks or Adults Manipulation of gonads in vivo Manipulation of spermatozoa Fig. Kino et al. Naito et al. but production of viable offspring derived from the donor blastodermal cells has not been successful. however. The injected quail blastodermal cells differentiate into various tissues and organs including gonads in the quail–chicken chimeras (Watanabe et al. In vitro fertilization or intracytoplasmic sperm injection will serve to clarify this issue. factors such as the size of the eggs. Animal Science Journal (2003) 74. Fertilized ova are obtained from the magnum of the oviduct 2. 1991c. To produce donor-derived offspring from interspeciﬁc germline chimeras. 1997. Harvey et al. the yolk is covered by a small capsule of thick albumen.75 h after the preceding egg is laid. DNA is injected into the germinal disc of the fertilized ovum near the female pronucleus. 2002). 3 Various approaches for DNA transfer into chickens. DNA TRANSFER INTO CHICKENS Various attempts have been made to introduce exogenous DNA into chickens.M. Transferred chicken PGC also settled in quail gonads (Ono et al. The manipulated ova are cultured until hatching. manipulation of blastodermal cells or primordial germ cells. 1994b. 157–168 162 . 1998). 1990). 3). in circular form. or manipulation of spermatozoa. This section describes various approaches to DNA transfer into chickens by non-viral methods (Fig. DNA in linear form injected into the germinal disc is ligated rapidly after injection to form random concatamers. Single-cell stage Microinjection of DNA into fertilized ovum Stage X Manipulation of blastodermal cells Interspeciﬁc avian chimeras Interspeciﬁc avian chimeras are very useful for proliferating endangered avian species and studying immune rejection of donor cells in the chimeras. 1998b) and chicken-speciﬁc DNA was observed in sperm samples from matured chimeras (Li et al. In other studies. The transferred quail PGC settled in the chicken gonads. 1992). At that time. Viable offspring derived from the donor PGC in interspeciﬁc chimeras have yet to be produced even by the transfer of PGC isolated from embryonic blood. 1991b).
Using a gene gun.0% at day 3 of incubation after the injection. The introduced DNA was found to be present in an episomal form and disappeared during the life span of the adult birds. The introduced DNA was expressed in chimeric embryos cultured for 2–3 days following injection (Etches et al. 1997. their ability to contribute to the germline of chimeric chickens was limited to the ﬁrst 7 days in culture. 1997b). Manipulation of primordial germ cells Manipulation of PGC could ensure the transmission of genetic modiﬁcations to the next generation via germline chimeric chickens. 1997b). and the DNA was transmitted to the offspring (Love et al. Putative pluripotent stem cells (ES cells) derived from stage X blastodermal cells have been identiﬁed and maintained in vitro in long-term culture (Pain et al. 2002). Dispersed blastodermal cells obtained from the area pellucida can be cultured for at least 48 h and are able to enter the germline of recipient embryos. Primordial germ cells obtained from the gonads of 5-day incubated embryos were cultured in vitro with stroma cells and were found to proliferate approximately 3 times over a few days. 1994). PGC cultured in vitro have been used. Their transfer from one embryo to another makes it possible to produce germline chimeric chickens. Fraser et al. 2000). and transferred to recipient embryos. Maruyama et al. Hatchlings were grown to maturity and exogenous DNA was shown to be present in their spermatozoa. the DNA disappeared from the chickens as they matured. Primordial germ cells were transfected in vivo by injecting a liposome-DNA mixture into the bloodstream of recipient embryos (Watanabe et al. Although the DNA was transmitted to the next generation. Offspring derived from the FACS-sorted blastodermal cells were efﬁciently obtained from the chimeric chickens. but most of the DNA was gradually lost during embryonic development. The chicken EG cells differentiated into various tissues in chimeric chickens when injected into a stage X blastoderm. 1993. 1998. but was reduced to 11. The ability of such cultured cells to contribute to the somatic and germline lineages has been found to be considerably reduced compared with freshly collected stage X blastodermal cells (Etches et al. Stage X blastodermal cells were efﬁciently transfected in vitro by lipofection or electroporation and injected into intact or compromised recipient embryos (Brazolot et al. 2000a) transfected PGC obtained from embryonic blood by lipofection and transferred them to recipient embryos. or the germline cells may be lost from the cell population during long- Animal Science Journal (2003) 74. 1996. term culture. 1991. Tajima et al. To select successfully transfected blastodermal cells. stage X blastodermal cells were cultured in vitro. 1992). Transfected blastodermal cells were FACSsorted. They may lose the ability to differentiate to the germline. Gene transfer into chickens using cultured blastodermal cells has not yet succeeded. 1999). Manipulation of blastodermal cells The stage X blastoderm consists of approximately 60 000 cells and contains approximately 30 PGC or their precursor cells.1% at hatching. but transfection efﬁciency was low. 2001). Freshly collected blastodermal cells were dissociated and transferred to recipient embryos of the same developmental stage to produce somatic and germline chimeric chickens (Etches et al. In order to achieve stable incorporation of exogenous DNA in chickens. The chicken EG cell colonies were uniformly round. 1999. These cultured PGC retained the ability to re-enter the gonads of recipient embryos and gave rise to viable offspring (Chang et al. the introduced DNA was not transmitted to the next generation. Although offspring derived from the transfected PGC were efﬁciently obtained from the germline chimeric chickens. 1995). suggesting that the DNA was episomal. The percentage of embryos with foreign DNA in the gonads was 95. (1998a. multilayered and well delineated. but germline contribution of these cells has not been proven. The circulating PGC were transfected and migrated to the germinal ridges. 1993.Manipulation of avian embryos Integration of the injected DNA into the host chromosomes was observed. Although these cells show features similar to mouse ES cells over a long period. PGC located in the germinal crescent region were transfected (Li et al. Han et al. 1996). Acloque et al. Naito et al. The integration efﬁciency was enhanced by use of the Drosophila transposable element mariner (Sherman et al. 157–168 163 . but the introduced DNA was not transmitted to the offspring (Speksnijder et al. The PGC isolated from the gonads of 5-day incubated embryos were cultured on a feeder layer over 4 months and established a chicken embryonic germ (EG) cell line (Park & Han 2000). The introduced DNA was efﬁciently expressed in the gonads of the recipient embryos. The introduced DNA was expressed in the PGC that settled in the gonads. The direct microinjection of DNA into the germinal disc is at present the only non-viral method that has successfully produced transgenic chickens. 1998).
the in vivo electroporation method is suitable for introducing exogenous DNA into a limited part of a targeted site in the embryos. 1993). Transfection of testes in live chickens by lipofection or electroporation seems to be a promising method for introducing exogenous DNA into spermatogonia. Sano et al. The transmitted DNA was mostly present episomally and disappeared during embryonic development. These techniques for the cryopreservation of germline cells make it possible to conserve genetic material in avian species and accelerate the production of transgenic chickens. cryopreservation of spermatozoa has already been achieved (Lake & Stewart 1978). (1994c) ﬁrst succeeded in preserving PGC isolated from embryonic blood in liquid nitrogen. 1997. but ova or fertilized eggs cannot be preserved in the same way because of their large size and yolk-laden structure. 1998. 1997. transfection efﬁciency was varied and a low expression rate of exogenous DNA in the embryonic tissues was observed. 157–168 164 . NAITO DNA transfer into chickens via PGC is one of the most promising methods. Transfection of a stage X blastoderm in vivo by electroporation made it possible to introduce exogenous DNA into early chicken embryos (Naito et al. 1998. When electric pulses were applied vertically to the blastoderm layer using CONCLUSION The advent of techniques to culture chicken embryos from the single-cell stage to hatching has accelerated Animal Science Journal (2003) 74. 1998. 2001b) would make it possible to produce transgenic chickens routinely in future. Naito et al. PRESERVATION OF AVIAN GENETIC RESOURCES Cryopreservation of germ cells from avian species conserves genetic material for the improvement of chickens and facilitates the preservation of endangered species (Naito 2003). 2000b. 1992. Recently. In chickens. Naito et al. 2002). When electric pulses were applied horizontally to the blastoderm layer using parallel type electrodes. 2000b). 1998b) or in vitro culture of PGC (Naito et al. novel electrodes. 2001c). Viable offspring were successfully produced from germline chimeric chickens produced by transfer of frozen-thawed blastodermal cells (Kino et al. Development of highefﬁciency transfection methods for PGC (Hong et al. However. This success may lead to the production of pharmaceutical proteins in eggs. The frozen stored PGC were capable of giving rise to viable offspring via germline chimeric chickens. spermatozoa with exogenous DNA will be continuously produced. In vivo electroporation and lipofection Recent development of lipofection and electroporation techniques enables us to transfect animal cells efﬁciently in vitro and in vivo (Muramatsu et al. Primordial germ cells obtained from the gonads of 5-day incubated embryos were also preserved in liquid nitrogen and were subsequently able to produce viable offspring via germline chimeric chickens (Tajima et al. If exogenous DNA can be introduced into the spermatogonia. 1998). and the lipofection method seems suitable for introducing exogenous DNA into a relatively broad area of the embryos (Muramatsu et al. 1997). Sperm microinjection into a newly ovulated ovum could be applied to introduce exogenous DNA into chickens. Naito et al. Petitte et al. and cryopreserved PGC can give rise to viable offspring via germline chimeric chickens. During the prepubertal phase. Naito et al. The in vivo electroporation method was applied to the oviduct of laying hens and resulted in synthesis of human erythropoietin protein in the oviduct cells (Ochiai et al. Blastodermal cells at stage X can be frozen in liquid nitrogen prior to their injection into recipient embryos to make chimeras (Naito et al. 2000b. efﬁcient transfection of PGC could be expected by in vivo electroporation applying electric pulses vertically to the blastoderm layer. Manipulation of spermatogonia and spermatozoa Spermatogonia are stem cells for producing spermatozoa. transfection efﬁciency was enhanced and a high rate of expression of exogenous DNA in the embryonic tissues was observed (Naito et al. As PGC are located in the ventral surface of the epiblast of the stage X blastoderm.M. spermatogonia increase in number through mitotic division and some of the spermatogonia are transformed into primary spermatocytes. 1998). 2003). When transfecting early chicken embryos. exogenous DNA has been transmitted to embryos by transfecting spermatozoa and artiﬁcially inseminating them into hens (Squires & Drake 1997). Cryopreservation of PGC provides an alternative means for conserving both male and female genetic material. linker based sperm-mediated DNA transfer has been reported in pigs and mice and the technique is applicable in chickens (Chang et al. 2002).
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