No abstract

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.

No abstract

Messenger RNA isolated from chicken pituitaries was used to construct a chicken pituitary cDNA library. A chicken growth hormone cDNA clone was isolated using 32P-labeled mammalian growth hormone cDNA probes. The amino acid sequence (derived from the DNA sequence) of the mature form of chicken growth hormone shows 77% homology with that of bovine growth hormone. The chicken growth hormone cDNA clone was used to generate a vector capable of producing chicken growth hormone in Escherichia coli. The recombinant E. coli-derived chicken growth hormone was similar to pituitary chicken growth hormone in several biochemical and immunological properties. The recombinant-derived hormone has been used to establish a sensitive radioimmunoassay for growth hormone determinations made from chicken sera. The chicken growth hormone gene has also been introduced into a retroviral vector capable of establishing productive infections of chicken cells both in in vitro and in vivo. The resulting infections are accompanied by the production of radioimmunoassay-detectable growth hormone. The concentrations of growth hormone in sera of Leghorn chickens infected with the recombinant retrovirus are three- to tenfold higher than in control animals.

Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5' long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2 -inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 104 G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 x 104 CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.

Chem. 272, 1-4). Therefore, we expressed both the wild type and the specific Cys to Ala form of chicken macrophage stimulating protein as recombinant proteins. After proteolytic activation, only conditioned media from COS cells transfected with the C665A chicken macrophage stimulating protein, but not from wild type chicken macrophagestimulating protein, or control vector, was detected by the Sea-immunoglobulin fusion protein in Western blotting experiments. Conditioned media containing the C665A chicken macrophage-stimulating protein readily caused Sea phosphorylation, while conditioned media containing the wild type chicken macrophage-stimulating protein was only effective at inducing receptor phosphorylation at high concentrations. In addition to receptor phosphorylation, the C665A chicken macrophage-stimulating protein induced phosphorylation of Shc, Erk1, and Erk 2. We conclude that macrophage-stimulating protein is a ligand of the Sea receptor protein-tyrosine kinase.The receptor protein-tyrosine kinases are a structurally related family of transmembrane proteins which regulate a wide variety of cellular responses to extracellular stimulation (2). The binding of ligand to the extracellular domain of these receptors results in rapid intracellular autophosphorylation followed by the phosphorylation of multiple downstream effector proteins (2).The Met receptor protein-tyrosine kinase (3, 4) is the prototype of a subfamily of receptor protein-tyrosine kinases. Other members of this subfamily include the RON 1 /Stk (5-7) and Sea receptors (8). A common feature of this subfamily is that the mature version of the receptor consist of a heterodimer between a small ␣ chain (ϳ35 kDa) and a larger ␤ chain (ϳ160 kDa) which arise from proteolytic cleavage of a precursor protein.

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 ...

No abstract