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.

A biologically active molecular clone of BALB/Moloney mink cell focusforming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the plSE peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses.

The mCAT-1 gene encodes a basic amino acid transporter that also acts as the receptor for murine ecotropic leukemia viruses. Targeted mutagenesis in embryonic stem cells has been used to introduce a germ-line null mutation into this gene. This mutation removes a domain critical for virus binding and inactivates amino acid transport activity. Homozygous mutant pups generated from these cells were -25% smaller than normal littermates, very anemic, and died on the day of birth. Peripheral blood from homozygotes contained 50% fewer red blood cells, reduced hemoglobin levels, and showed a pronounced normoblastosis. Histological analyses of bone marrow, spleen, and liver showed a decrease in both erythroid progenitors and mature red blood cells. Mutant fetal liver cells behaved normally in in vitro hematopoietic colony-forming assays but generated an anemia when transplanted into irradiated C.B.-17 SCID mice. Furthermore, reconstitution of the white cell compartment of SCID mice by mutant fetal liver cells was less complete than that observed with a mixed population of wild-type and heterozygous fetal liver cells. Primary embryo fibroblasts from mutant mice were completely resistant to ecotropic retrovirus infection. Thus, mCAT-1 not only appears to be the sole receptor for a group of murine ecotropic retroviruses associated with hematological disease but also plays a critical role in both hematopoiesis and growth control during mouse development.

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.