Development of transgenic chickens expressing bacterial β‐galactosidase

Abstract

Replication-defective retroviral vectors are efficient vehicles for the delivery of exogenous genes, and they may be used in the generation of transgenic animals. The replication-defective retroviral SNTZ vector carrying the lacZ gene with a nuclear localized signal was injected into the subgerminal cavity of freshly laid eggs. Subsequently, the eggs were allowed to hatch, and the chickens were screened for the lacZ gene by using the polymerase chain reaction. Eight of 15 male chickens that survived to sexual maturity contained the lacZ gene in their semen. Subsequently, these males were mated with wild-type female chickens. From one of the eight lacZ-positive G 0 males, two lacZ-positive male chickens were produced from a total of 224 G 1 progeny for a germline transmission rate of 0.89%. Both G 1 male chickens carrying the lacZ gene were mated with wild-type female chickens and 46.5% of the G 2 progeny contained the lacZ gene, which is consistent with the expected Mendelian 50% ratio for a heterozygous dominant allele. The product of the lacZ gene, nuclear localized ␤-galactosidase, was expressed in primary myoblast cultures derived from G 2 chickens, and it was also expressed in whole G 2 chicken embryos. Developmental Dynamics 226:439 -445, 2003.

Status of transgenic chicken models for developmental biology

“…The modifications of the original procedures of Perry (1988) and Rowlett and Simkiss (1987) by Borwornpinyo (2000) resulted in 20% to 35% hatchability when used in conjunction with retroviral injections. The hatchability was sufficient to generate G0 roosters carrying the lacZ gene in their semen (Mozdziak et al, 2003). Currently, the windowing and the ex ovo culture are the only ways to introduce foreign DNA into a chicken embryo, and have viable offspring.…”

“…Retroviral gene transfer techniques have been used the most frequently to generate transgenic chickens (Bosselman et al, 1989a(Bosselman et al, ,b, 1990Harvey et al, 2002a,b;Harvey and Ivarie, 2003;McGrew et al, 2003;Mozdziak et al, 2003), because high retroviral titers make it possible to transfer the gene of interest to many cells. Briefly, retroviruses have an RNA genome encased in a protein core containing integrase, reverse transcriptase, and protease.…”

“…The work of Harvey et al (2002a,b) demonstrated that a chicken can be engineered to express a foreign protein in the egg showing the feasibility of the hen as a bioreactor for the production of therapeutic proteins. Second, a new chicken model for developmental biology research has been currently engineered (Mozdziak et al, 2003) by using a retroviral vector system that has been previously shown to have high expression in the chicken (Mikawa et al, 1991(Mikawa et al, , 1992. These transgenic chickens are a new model system for developmental biology research (Mozdziak et al, 2003) that is an improvement over the quail:chick chimera, because the chickens carry the lacZ gene and express betagalactosidase, making it possible to track cells into a wild-type chick embryo (Giamario and Mozdziak, unpublished observations).…”

“…Subsequently, the investigators must have sufficient space to maintain flocks for test-mating the birds. Despite these challenges, the recent successes of Harvey et al (2002a,b;2003), McGrew et al (2003), and Mozdziak et al (2003) show that transgenic chicken technology can be used to generate tools for biological research.…”

Somatic transgenesis using retroviral vectors in the chicken embryo

“…The SNVbased vectors have been used in variety of embryonic chick tissues, such as the blastoderm, the primitive streak, the somitic and somatic mesoderm, epithelial heart tube, neural crest, vascular precursors, limb bud, central nervous system, and kidney primordium (Mikawa et al, 1992a,b;Mikawa and Fischman, 1992;Herzlinger et al, 1992;Epstein et al, 1994;Mima et al, 1995;Gourdie et al, 1995;Cohen-Gould and Mikawa, 1996;Mikawa and Gourdie, 1996;Itoh et al, 1996;Ong et al, 1998;Hyer et al, 1998Hyer et al, , 1999Cheng et al, 1999;Wei and Mikawa, 2000;Takebayashi-Suzuki et al, 2000;Das et al, 2000;Hatcher et al, 2001;Kanzawa et al, 2002;Ballard and Mikawa, 2002;Sugi et al, 2003). It has been shown that CXL and SNTZ are capable of infecting embryos as early as the prestreak stages (Wei and Mikawa, 2000;Mozdziak et al, 2003) and that the LacZ activity becomes detectable within 12-13 hr after infection (Wei and Mikawa, 2000). Coinjection of CXL and SNTZ has been used to assess clonal boundaries in lineage studies in the heart (Mikawa et al, 1992b).…”