Difficulties associated with in vitro manipulation and culture of the early chicken embryo have restricted generation of transgenic chickens to approaches that use replication-competent retroviruses. The need to produce transgenic chickens in the absence of replicating virus prompted development of a new method of gene transfer into the chicken. Microinjection of the replication-defective reticuloendotheliosis virus (REV) vector ME111 beneath unincubated chicken embryo blastoderms results in infection of germline stem cells. This vector contains genetic information exogenous to the chicken genome, including both the herpes simplex virus type 1 thymidine kinase gene and the Tn5 neomycin phosphotransferase gene. About 8 percent of male birds hatched from injected embryos contained vector DNA in their semen. All four positive males tested passed vector sequences onto their progeny. Analysis of G1 offspring showed that gonads of G0 male birds were mosaic with respect to insertion of vector provirus. Thus, primordial germ cells present in the unincubated chicken embryo blastoderm are susceptible to infection by defective REV vectors.

This report describes expression of heritable reticuloendotheliosis virus (REV) vector ME111 in 20 independent lines of transgenic chickens. The results are strikingly different from studies of Moloney virus in transgenic mice, where restricted expression of inherited proviruses has led to their use primarily as insertional mutagens rather than general agents for gene transfer. In contrast, the REV ME111 provirus is actively transcribed in a variety of tissues from transgenic chickens, is expressed from transcriptional control elements present in the long terminal repeat of the provirus, and codes for active neomycin phosphotransferase II. The REV vector system as applied to the chicken represents a departure from the long-established paradigm of retroviral transgenes in mice and provides a new approach to the study of avian biology.Replication-defective vectors derived from reticuloendotheliosis virus (REV) can infect pluripotent stem cells when injected beneath the blastoderm of unincubated chicken eggs (1, 2). Gene transfer at this stage of embryonic development requires the high efficiency of viral infection because the blastoderm contains many thousands of cells (3,4). This procedure gives rise to mosaic chickens which upon subsequent breeding yield transgenic animals hemizygous for unique provirus insertions. Although these viral transgenes are expressed in somatic cells ofthe infected embryo, a major question has been whether inherited provirus would remain transcriptionally active.The first germ-line insertion of experimentally introduced provirus was achieved by infection of the early mouse embryo (5). This and other studies have shown that provirus passed through the mouse germ-line is often transcriptionally inactive (6)(7)(8)(9)(10)(11)(12). The ability to manipulate the mouse embryo has led to alternative methods of gene transfer such as nuclear injection of cloned DNA (13,14) (Fig. la). As a positive control duplicate filters were hybridized with a 1.1-kb Pst I fragment derived from the plasmid ptl, which contains an a-tubulin cDNA homologous with the five-member a-tubulin gene family (24,25). RNA Dot Blot Analysis. Total RNA was denatured in formaldehyde and immobilized on GeneScreenPlus membranes (New England Nuclear). Duplicate RNA samples were treated with 2 M NaOH at 65TC as a control for DNA contamination (data not shown). Filters were baked 60 min at 80TC and prehybridized at 650C for 1 hr in 1 M sodium phosphate, pH 7.2/0.5% SDS/0.1% Ficoll/0.1% polyvinylpyrrolidone/0.1% bovine serum albumin/1 mM EDTA containing 50 gg of denatured salmon sperm DNA per ml (22). A DNA probe (ME111 or a-tubulin; 5 x 106 cpm) was added and filters were hybridized overnight at 650C. Filters were washed twice in 15 mM NaCI/1.5 mM sodium citrate, pH 7/0.2% SDS for 60 min at 65TC and subjected to autoradiography.Northern Blot Analysis. Total RNA was passed once over a Stratagene "quick push" oligo(dT)-cellulose column according to the manufacturer's instructions. Two hundred nanograms of RNA from the ME111 ...

Replication-defective vectors derived from reticuloendotheliosis virus were used to transduce exogenous genes into early somatic stem cells of the chicken embryo. One of these vectors transduced and expressed the chicken growth hormone coding sequence. The helper cell line, C3, was used to generate stocks of vector containing about 104 transducing units per ml. Injection of 5to 20-,ul volumes of vector directly beneath the blastoderm of unincubated chicken embryos led to infection of somatic stem cells. Infected embryos and adults contained unrearranged integrated proviral DNAs. Embryos expressed the transduced chicken growth hormone gene and contained high levels of serum growth hormone. Blood, brain, muscle, testis, and semen contained from individuals injected as embryos contained vector DNA. Replication-defective vectors of the reticuloendotheliosis virus transduced exogenous genes into chicken embryonic stem cells in vivo.