Germline transmission of exogenous genes in the chicken.

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.

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

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 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) 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.

Streptomycin: separation of polysomal and non-polysomal messenger ribonucleoproteins.

In the presence of 0.02 M streptomycin, all of the polysomes precipitate from male cricket (Acheta domesticus) accessory gland and chick embryonic tissue post-mitochondrial fractions. All non-polysomal messenger-like ribonucleoprotein preparations tested remain in solution.

Genome organization of retroviruses. V. In vitro-synthesized Moloney murine leukemia viral DNA has long terminal redundancy.

Purified virions of Moloney murine leukemia virus can synthesize genome-length double-stranded DNA in vitro. Two predominant species of long DNA transcripts, with average sizes of 9.1 and 8.5 kilobases (kb) can be identified. Both species of DNA contain the negative (complementary to viral RNA) and positive (same polarity as viral RNA) strands. However, only the negative strand of the 8.5-kb species can be identified if the synthesis of DNA is carried out in the presence of the drug actinomycin D. The 9.1-kb species appears to be slightly larger than the genomic RNA. If the linear double-stranded 9.1-kb species is treated with Escherichia coli exonuclease III and allowed to anneal, circular DNA molecules can be observed. Furthermore, polyadenylate-containing short genomic RNA fragments (0.5 to 1.0 kb) can anneal to both the 5' and the 3' termini of 9.1-kb complementary DNA. The polyadenylate moiety of the RNA fragments can be identified by tagging it with circular polyoma DNA containing polydeoxybromouridylic acid tails. Thus, the 9.1-kb complementary DNA transcript with two circular polyoma DNA molecules at its termini can be observed. However, when similar annealings are performed with 8.5-kb complementary DNA species, only one end of the resulting molecule has circular polyoma DNA. We conclude that the 9.1-kb complementary DNA species has a large terminal redundancy. The sequences involved in terminal redundancy appear to be derived from the 3' end of the genomic RNA.

Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA.

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (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 p15E 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.

Genome organization of retroviruses. VI. Heteroduplex analysis of ecotropic and xenotropic sequences of moloney mink cell focus-inducing viral RNA obtained from either a cloned isolate or a thymoma cell line.

The genome of a recombinant murine leukemia virus capable of inducing focal areas of morphological alteration in mink lung fibroblasts was studied by heteroduplex analysis. The dual-tropic recombinant virus was isolated from a thymoma cell line (Th16.3) and is referred to as BALB/Moloney mink cell focus-inducing virus (BALB/Mo-MCF virus). The nucleic acid sequences of RNA from virions obtained from either a thymoma cell line (Th16.3) or a clonal isolate (BALB/Mo-MCF81) were compared with the genomes of ecotropic and xenotropic viruses. The following inferences were drawn (i) A single nonhomologous region (substitution loop alpha) of about 0.7 kilobase was observed in a heteroduplex formed between Moloney murine leukemia virus complementary DNA (cDNA) and BALB/MoMCF81 RNA. This nonhomology region was mapped between 1.71 and 2.40 kilobases from the 3' end of the genome. (ii) The predominant class of heteroduplexes formed between virion RNA obtained from the thymoma cell line (Th16.3) and Moloney murine leukemia virus cDNA showed a substitution loop similar to that observed with the RNA obtained from a cloned isolate, BALB/Mo-MCF81. However, there were other molecules with additional regions of nonhomology. (iii) Heteroduplexes formed between NZB xenotropic RNA and ecotropic Moloney murine leukemia virus cDNA exhibited four major nonhomology regions extending 0.75 to 1.46, 2.0 to 2.8, 3.6 to 4.3, and 7.4 to 7.9 kilobases from the 3' end of the genome. (iv) The MCF-specific substitution loop alpha (1.71 to 2.40 kilobases) appeared as a duplex region when NZB xenotropic RNA was hybridized to cDNA transcripts synthesized by virions obtained from thymoma cell line Th16.3. The position of the other substitution loops observed in a heteroduplex formed between NZB xenotropic RNA and Moloney murine leukemia virus cDNA was not affected. (v) Heteroduplexes formed between xenotropic BALB virus 2 cDNA and NZB xenotropic RNA demonstrated a large degree of nucleic acid sequence homology. Of the 29 heteroduplexes examined, 24 appeared to be homoduplexes, and in the remaining 5 heteroduplexes only one region of nonhomology located between 3.2 and 3.8 kilobases from the 3' end of the genome could be identified. Hybridization of BALB virus 2 xenotropic RNA to NZB xenotropic cDNA followed by digestion with single-strand-specific nuclease S1 showed an 80% sequence homology.

Increased expression of p53 protein in human leukemia cells.

We examined synthesis of the cellular phosphoprotein p53 in fresh bone marrow or peripheral blood cells from normal donors and from patients with leukemia, preleukemia, or other hematopoietic disorders. Lysates of cells labeled with [35S]methionine were immunoprecipitated with monoclonal antibodies to p53, and the immunoprecipitates were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Bone marrow or peripheral blood cells from 8 of 33 patients with hematopoietic disorders showed increased p53, seven of the eight occurring in cells of patients with preleukemia or acute myelogenous leukemia. Increased p53 synthesis was not associated with p53 gene amplification, as shown by Southern blot analysis. Synthesis of p53 was not increased in any of nine normal human bone marrow samples or eight normal human peripheral blood granulocyte, macrophage, and lymphocyte samples. The hematopoietic cells of patients in remission or with chronic forms of leukemia did not generally synthesize elevated levels of p53. In addition, we found negligible p53 mRNA and protein expression in a variety of human myeloid leukemia lines blocked at different stages of differentiation. Southern blot analysis showed that, except for the HL-60 cells, the p53 gene of the myeloid cell lines was intact. In view of recent evidence implicating p53 in transformation of cultured cells, our results using fresh leukemia cells suggest that p53 may contribute to the phenotype of certain leukemias in vivo.

Molecular cloning of unintegrated Moloney mouse sarcoma virus DNA in bacteriophage lambda.

The covalently closed circular forms of unintegrated viral DNA obtained from cells infected with Moloney mouse sarcoma virus was cloned in bacteriophage lambda. The viral DNA was cleaved with restriction endonuclease HindIII and inserted in the unique HindIII site of lambda Charon 21A DNA. Recombinant clones containing virus-reactive DNA sequences were analyzed by restriction endonuclease mapping, R-loop formation, and infectivity assays. Two of eight genome-length recombinant clones characterized contained the large terminal repeat. Only the recombinant clones containing the large terminal repeat were able to induce focus formation in uninfected mouse fibroblasts.

Identification and molecular cloning of Moloney mouse sarcoma virus-specific sequences from uninfected mouse cells.

When uninfected mouse cell DNA is cleaved with restriction endonuclease EcoRI, a DNA fragment of 14.0 kilobases can be identified by hybridization to cloned DNA containing sarcoma specific sequences of Moloney mouse sarcoma virus (M-MSVsrc). The cellular DNA fragment contains the entire M-MSVsrc specific sequences. The 14.0-kilobase EcoRI DNA fragment was cloned in bacteriophage lambda. The sequence organization of a recombinant clone, lambda . MTX-1, was analyzed by restriction endonuclease mapping, nuclease S1 mapping, and electron microscopy. The results indicate that lambda . MTX-1 contains an uninterrupted stretch of 1.0 kilobase similar to that found in the M-MSV genome.

Ribosomal proteins in normal simian cells, SV40-transformed simian cells, and simian cells infected with SV40, adenovirus 5, and vesicular stomatitis virus.

The protein patterns of monosomes and polysomes isolated from the T-22 line of SV40-transformed GMK cells and from uninfected CV-1 cells and CV-1 cells infected with SV40, adenovirus 5, or vesicular stomatitis virus were analyzed by two-dimensional PAGE. All gel patterns were similar except for the presence of one additional protein associated with T-22 monosomes.

Heritable retroviral transgenes are highly expressed in chickens.

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 of reticuloendotheliosis virus transduce exogenous genes into somatic stem cells of the unincubated chicken embryo.

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 10(4) transducing units per ml. Injection of 5- to 20-microliters 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.

Anemia and perinatal death result from loss of the murine ecotropic retrovirus receptor mCAT-1.

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 approximately 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.

Molecular cloning of unintegrated and a portion of integrated moloney murine leukemia viral DNA in bacteriophage lambda.

A covalently closed circular form of unintegrated viral DNA obtained from NIH 3T3 cells freshly infected with Moloney murine leukemia virus (M-MLV) and a port of the endogenous M-MLV from the BALB/Mo mouse strain have been cloned in bacteriophage lambda. The unintegrated viral DNA was cleaved with restriction endonuclease HindIII and inserted into the single HindIII site of lambda phage Charon 21A. Similarly high-molecular-weight DNA from BALB/Mo mice ws cleaved sequentially with restriction endonucleases EcoRI and HindIII and separated on the basis of size, and one of the two fractions which reacted with an M-MLV-specific complementary DNA was inserted into the HindIII site of Charon 21A. Recombinant clones containing M-MLV-reacting DNA were analyzed by restriction endonuclease mapping, heteroduplexing, and infectivity assays. The restriction endonuclease map of the insert derived from unintegrated viral DNA, lambda x MLV-1, was comparable to published maps. Electron microscope analysis of the hybrid formed between lambda x MLV-1 DNA and 35S genomic M-MLV RNA showed a duplex structure. The molecularly cloned lambda x MLV-1 DNA contained only one copy of the long terminal repeat and was not infectious even after end-to-end ligation of the insert DNA. The insert DNA derived from endogenous M-MLV, lambda x MLVint-1, contained a DNA stretch measuring 5.4 kilobase pairs in length, corresponding to the 5' part of the genomic viral RNA, and cellular mouse DNA sequences measuring 3.5 kilobase pairs in length. The viral part of the insert showed the typical restriction pattern of M-MLV DNA except that a single restriction site, PvuII, in the 5' long terminal repeat was missing. Reconstructed genomes containing the 5' half derived from the integrated viral DNA and the 3' half derived from the unintegrated viral DNA were able to induce XC plaques after transfection in uninfected mouse fibroblasts.

Transmission of exogenous genes into the chicken.

Injection of infectious non-replicating REV vector directly beneath the chicken blastoderm leads to infection of embryonic stem cells. Vector sequences are present in a variety of specialized tissues of embryos and mature birds derived from infected blastoderms. Breeding studies show that replication-defective REV vectors can transfer heritable, non-viral genetic information into the chicken germ line.