Consistent production of transgenic chickens using replication-deficient retroviral vectors and high-throughput screening procedures.

We have developed a novel method of DNA extraction combined with a high-throughput method of gene detection allowing thousands of potentially transgenic chicks to be screened quickly and reliably. By using this method and a replication-deficient retroviral vector based on avian leukosis virus (ALV), we have demonstrated germline transmission of three different transgenes. Several generations of chickens carrying intact transgenes were produced, validating the use of the ALV retroviral vectors for large-scale production of transgenic flocks. Fourth-generation chicks that were nontransgenic, hemizygous, or homozygous for the transgene were identified with the combined genetic screening methods.

Monitoring Mycoplasma gallisepticum and Mycoplasma synoviae infection in breeder chickens after treatment with enrofloxacin.

Three experimental strains of breeder chickens were accidentally exposed to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), presumably from a newly introduced group of leghorn-type pullets. The experimental strains subsequently became infected and were diagnosed positive for MG and MS by the serum plate agglutination (SPA) test and confirmed by the hemagglutination inhibition (HI) test and the polymerase chain reaction (PCR) of tracheal swabs. Treatment with 10 mg/kg enrofloxacin via drinking water for 14 days was elected. Before and after initiation of treatment, MG and MS were monitored for changes by SPA, HI, PCR, and culture, with sampling intervals ranging from 1 wk to 7 wk. MG and MS SPA, HI, PCR, and culture were performed at each sampling period, with the exception of weeks 1.0 and 6.5. Week 1.0 included SPA and His for MG and MS. Week 6.5 included PCR and culture for MG and MS. The MG and MS SPA results were positive throughout the 29-wk trial period. MG HI titers declined until the last sampling, whereas the MS HI titers did not decline significantly. PCR for MG yielded only one positive result, which occurred before treatment. MS PCR remained positive throughout the trial period. MG was never isolated from any sample; however, one MS organism was isolated during treatment. The treatment regimen was effective for MG on the basis of PCR results. Treatment with enrofloxacin did not eliminate SPA reactions during the 29-wk trial period. MG HI titers remained in the suspicious range throughout the remainder of the trial period. Four weeks after the treatment ended, MG HIs were reduced by approximately 40%, with MS HIs remaining high throughout the 29-wk period. PCR appeared to be a sensitive and specific test on the basis of correlation with HIs. On the basis of the isolation of MS during treatment and continued subsequent PCR positive reactions, the treatment for MS with enrofloxacin was not as efficacious as for MG.

A modified method of shell windowing for producing somatic or germline chimeras in fertilized chicken eggs.

Stage X chick blastoderms following oviposition were accessed via a small window in the egg. Windowing, however, substantially reduces the hatchability of eggs containing early embryos. For example, only 32 of 389 (8.2%) eggs hatched after standard windowing with or without irradiation or injection. Ex ovo culture systems can overcome this problem but are labor intensive. A modification of a standard windowing technique has yielded an average hatch rate of 32% for 892 windowed eggs independent of incubator type, gamma-irradiation, or injection of the embryo. This was a fourfold increase over a standard windowing method. Similar hatch rates were observed using fertile eggs from five chicken lines [Barred Plymouth Rock (BR), Athens-Canadian (AC), Line 0, SPAFAS, and commercial White Leghorns (WL)]. The modification involves covering the egg shell membrane with PBS after grinding away the shell and before piercing the membrane. The window is then sealed by overlaying with fresh shell membrane and cementing it in place once it has dried. The method has been used successfully for the production of somatic and germline chimeras because donor BR blastodermal cells injected into Stage X, gamma-irradiated recipient embryos from WL or AC yielded a hatch of 33.7%, of which 42.3% were feather chimeras. Two of 11 cockerels tested were germline mosaics bearing at least 1% BR sperm. The modified windowing technique may be broadly applicable in emerging technologies in avian transgenesis and development.

Manipulation of blastodermal cells.

Blastodermal cells isolated from newly laid, unincubated eggs are virtually uncommitted cells that exhibit many of the properties of pluripotential stem cells. They can be transferred from donor to recipient embryos and contribute to both somatic tissues and the germline. Blastodermal cells that have been maintained in culture for 7 d express the epitopes ECMA-7 and SSEA-1, which are also expressed by mouse embryonic stem cells. After culture for up to at least 7 d, blastodermal cells retain the ability to differentiate into somatic tissues and the germline both in vivo and in vitro. Proliferation in the absence of differentiation of blastodermal cells is stimulated by the presence of Leukemia Inhibitory Factor (LIF) and other ligands that interact with the gp130 receptor, and differentiation is stimulated by exposure to retinoic acid. Blastodermal cells also possess high levels of telomerase activity, which is shared by immortalized cells and cells within the germline. Blastodermal cells can be transfected and will express foreign genes both in vivo and in vitro. Transfected cells can be isolated by fluorescence activated cell sorting and can be cryopreserved without losing their ability to contribute to either somatic tissues or the germline. These properties of blastodermal cells make them ideal vectors for introducing genetic modifications to the germline.