Robust and ubiquitous GFP expression in a single generation of chicken embryos using the avian retroviral vector, RCASBP
Introduction
The chicken embryo is a widely used model in developmental biology. Large numbers of fertile eggs are readily available and easy to maintain, embryos can be accessed during development, and chicken embryology is well documented (Brown et al., 2003). In addition, the chicken genome has now been sequenced (Hillier, 2004; Dequeant and Pourquie, 2005). Functional genomics in the avian model, however, has lagged behind that of mammals, and the production of transgenic birds has proved technically difficult (Mozdziak and Petitte, 2004). This is partly due to the features of avian reproductive physiology. At the time of fertilization, pronuclei in the chicken egg are difficult to detect on a large yolky oocyte. Up to 30 male pronuclei can be present on the germinal disc, while the ovum becomes surrounded by a large volume of albumen, and subsequently a shell membrane and eggshell, making access difficult (Thorne, 1995). This has resulted in the targeting of alternative developmental stages for genetic manipulation: either primordial germ cells isolated from developing embryos or the freshly laid blastoderm (Vick et al., 1993a, Vick et al., 1993b; Perry and Sang, 1993). A number of studies have focussed on the isolation and genetic modification of primordial germ cells from early embryos (Naito et al., 1998, Naito et al., 1999; Mozdziak et al., 2005; Chang et al., 1997; Park and Han, 2000; Park et al., 2003a, Park et al., 2003b; Hong et al., 1998; van de Lavoir et al., 2006). The aim of these approaches is to introduce modified germ cells into host embryos, where they contribute to the germline, leading to chimeric hatchlings (Han et al., 2002; Harada et al., 1993; Naito et al., 1994; van de Lavoir et al., 2006). This method requires the efficient isolation of germ cells from donor embryos, transfection of DNA in vitro, and successful introduction into embryos that have been depleted of endogenous germ cells. The rate of germline transmission using this method can be quite low, and current approaches involve breeding chimeric birds.
A more direct approach involves the use of viral vectors delivered directly into early blastoderms. In these studies, viruses carrying reporter genes such as GFP or LacZ stably integrate into the genome of the embryo, yielding chimeras that are hatched and screened at sexual maturity for somatic and germline transgenesis (Koo et al., 2006; Kwon et al., 2004; Mozdziak et al., 2003). Germline transgenic birds are then bred through G1 or G2, until homozygosity and ubiquitous transgene expression (McGrew et al., 2004; Chapman et al., 2005; Ishii et al., 2004). Different retroviral vectors have been used to produce transgenic birds, including those generated from avian leukosis virus, reticuloendotheliosis virus (REV), spleen necrosis-derived (SNTZ) and Moloney murine leukemia viruses (MoMLV) (Kwon et al., 2004; Bosselman et al., 1989a, Bosselman et al., 1989b; Mozdziak et al., 2003; Salter et al., 1987a). These viruses have generally been detectable at low levels in the soma and germline of founder (G0) birds (reviewed in Sang, 2004). More recently, lentiviral vectors have gained popularity. Lentiviral vectors have been used to produce GFP transgenic mice, rats and other mammals (Lois et al., 2002; Hofmann et al., 2003). In chickens, ubiquitous GFP expression in G1 and G2 embryos has been achieved using a lentiviral vector containing the constitutive phosphogylcerol kinase promoter (Chapman et al., 2005). Lentiviral vectors have also been used to direct tissue-specific gene expression in avian embryos. For example, Scott and Lois (2005) generated G1 transgenic quail embryos with GFP expression restricted to neural tissue by using lentivirus with a human synapsin (neural) promoter.
All currently published methods of producing transgenic chicken embryos have reported mosaic expression of virus in the founder (G0) embryos, necessitating the breeding of chimeras. This is due to the nature of the retroviral vectors used; lentivirus is replication incompetent, such that it infects an initial pool of cells but cannot spread horizontally to neighbouring cells. This restricts initial infection of the founder (G0) embryos, including low-level infection of germ cells. Here, we report the robust and ubiquitous expression of eGFP in a single (G0) generation of chicken embryos using Replication-Competent Avian sarcoma-leukosis virus, with a Splice acceptor, Bryan RSV Pol (RCASBP) (Hughes et al., 1987; Boerkoel et al., 1993; Hughes, 2004). RCASBP virus has been widely used to mis-express genes in specific embryonic structures such as the limb (Logan and Tabin, 1998), but it has not hitherto been shown to induce global transgene expression in chicken embryos. The advantage of RCASBP is that it is replication competent, spreading horizontally to neighbouring cells after infection, as well as vertically to daughter cells. The result is global transgene expression among a high percentage of directly infected embryos (over 80%), without the need to breed chimeric birds. The method produces a higher rate of global GFP expression among a population of embryos than other techniques. This transgenic model will be useful for developmental studies, particularly in tissues that have been difficult to transfect with exogenous DNA. However, in contrast to the soma, the germ cells were found to be GFP negative in infected embryos. This may allow the efficient isolation of germ cells through negative selection.
Section snippets
Propagation of RCASBP virus
GFP transgenic chicken embryos were produced by infection with Replication-Competent Avian Leucosis Sarcoma virus long terminal repeat (LTR) with Splice acceptor, Bryan RSV pol gene and subgroup B env gene, carrying enhanced Green Fluorescent Protein (RCASBP.B.eGFP). RCASBP.A.eGFP proviral DNA was a gift from Cliff Tabin (Harvard University, USA). The eGFP was excised from RCASBP.A and cloned into B strain (RCASBP.B), which was found to induce more widespread expression than A strain. As for
eGFP expression in transgenic embryos
Infection of susceptible embryos (SPAFAS C/E) with RCASBP.eGFP resulted in robust and ubiquitous GFP expression within a majority of embryos. Embryos injected with virus at the blastodermal stage (day 0; stage X) were examined at day 1, day 3.5 (developmental stage 19), day 5.5 (stage 28), day 8.5 (stage 35), and day 12.5 (stage 38). Almost all embryos examined at these stages (over 95%) showed some degree of eGFP expression, while an average of 80% showing global expression (Table 1). Robust
Discussion
Since it was developed some 20 years ago, the RCAS avian retrovirus has been used extensively as a vector for localised mis-expression of genes in embryonic chicken tissues (Hughes et al., 1987; Morgan and Fekete, 1996; Fekete and Cepko, 1993; Hartmann and Tabin, 2001). The study described here is the first to report the use of this vector to produce global transgene expression in avian embryos. Using RCASBP as a vector, we have achieved widespread GFP expression within a single generation of
Acknowledgements
We thank Dr. Helen Sang, Dr. Simon Lilico, Dr. Mike Clinton and Dr. Mike McGrew at the Roslin Institute, the United Kingdom, for demonstrating viral injections and embryo manipulation. Professor Cliff Tabin (Harvard Medical School) provided the viral vectors and Dr. Richard Pearce (Harvard Medical School) provided helpful advice. This work was supported by an Australian Research Council grant to CAS and AHS.
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