Genetic analysis of the growth curve of Rous sarcoma virus-induced tumors in chickens.

White Leghorn chicks homozygous for B19 MHC haplotype were selected for 18 generations on tumor regression after inoculation in the wing web with an SR-D strain of Rous sarcoma virus (RSV) at 4 wk of age. Each chick was assigned a tumor profile index (TPI) based on age at death and size of the tumor. During 18 generations, 2,010 birds were divergently selected on TPI for either progression or regression of the tumor (P and R lines). A Brody growth curve was fitted for each bird. Brody function parameters included the asymptotic tumor volume (A), the factor for increased growth in progression phase (K1), the factor for decreased growth in regression phase (K2), age at maximum volume (Tmax), and maximum volume of the tumor (Vmax). Tumor growth curves were found to be different according to line, sex, and restriction fragment pattern Y complex Rfp-Y MHC haplotype (Yw*15, Yw*16, and Yw*17). Within the P line, birds from the Yw*16 haplotype reached Vmax at an earlier age than Yw*15 and Yw*17, but with a lower Vmax value. Within the R line, tumor growth curves of birds from Yw*16 and Yw*17 haplotypes were similar. Rank correlations between the different parameters and TPI were low (between -0.26 and 0.36). Heritability estimated by the sire component was high for Vmax (0.73). Heritabilities of Tmax and K2 were moderate (0.20 to 0.23 for Tmax and 0.18 to 0.21 for K2) allowing these traits to be used as selection criteria. Heritabilities of A and K1 were lower than 0.12. Modeling the growth curve should contribute to better distinction between progressors and regressors.

Evolution of preproinsulin gene in birds.

The coding region of the preproinsulin gene has been cloned and partly sequenced in a variety of marine and terrestrial birds (28 species). All genes showed the "ancestral" structure with a large intron-2. The size of intron-2 changed considerably during the evolution of birds (2.4-4.2kb). The hydrophobicity of signal peptides was conserved. Bird C-peptides were predicted to be 28 amino acids long, but circulating C-peptides would be only 26 amino acids long, with Passer as a possible exception. Bird C-peptides were found to lack the sequences identified in mammals as responsible for peptide bioactivity and the structure of the central part. In contrast, predicted insulin sequences were highly conserved. Only two types of analog were identified: the hypoactive form (GluA8), present only in Anseriformes and the hyperactive form (His A8), present in all other species. Based on 3'-nucleotide sequence analysis (extending into intron-2), birds appeared to be monophyletic. Five groups were clearly identified: Paleognathae, Galliformes, Anseriformes, Passeriformes, and Charadriiformes. Paleognathae were suggested as the basal group, supporting the traditional view of avian evolution. Subsequent branching identified a gallo-anserae group and a group containing all other Neognathae. Surprisingly, Columba livia (Columbiforme order) clustered with Galliformes. With represented species, Procellariiformes and possibly Ciconiiformes, and Pelicaniformes were suggested as paraphyletic, in agreement with conclusions from some studies based on mitochondrial DNA sequences.

Alterations of the MDV oncogenic regions in an MDV transformed lymphoblastoid cell line.

AIMS: Lymphoblastoid cell lines derived from Marek's disease virus (MDV) induced tumours have served as models of MDV latency and transformation. They are stable and can be cultured with no detectable MDV genomic alterations upon repeated passaging. An MDV transformed lymphoblastoid T cell line (T9 cell line) has been reported to contain a disrupted MDV BamHI-H fragment and a Rous associated virus insertional activation of the c-myb protooncogene. In an attempt to define the respective participation of c-myb and MDV in the transformed phenotype of T9 cells, an analysis of MDV oncogenic sequences (BamHI-H, BamHI-A, and EcoQ fragments) was performed in these cells. METHODS: Using two different passages of the T9 cell line (late and early passages), the organisation of the MDV oncogenic regions and their expression in these cells were analysed. In vivo assessment of the oncogenicity of the virus contained within these cells was assessed by injecting them into 1 day old chickens. RESULTS: In T9 cells maintained in culture for up to six months (late T9), the MDV ICP4 gene was disrupted, whereas the meq gene was actively transcribed. The alterations of the MDV genome in these cells correlated with the inability of the virus to induce the classic signs of Marek's disease in 1 day old chickens. However, early T9 cells submitted to a limited number of passages induced classic MDV pathogenicity, as efficiently as the MDV control cell line (T5), and did not show gross structural changes in the oncogenic MDV sequences. CONCLUSIONS: Although the expression pattern of the MDV oncogenes in early T9 cells was identical to the one reported for other MDV transformed cells, longterm culture of an MDV transformed cell line containing a RAV insertional activation of the c-myb protooncogene led to the disruption of the MDV BamHI-H and BamHI-A oncogenic regions. In the late T9 cells MEQ was the only detected MDV oncoprotein. These results suggest that in the late T9 cells the truncated MYB protein compensates for the loss of MDV oncoproteins and reinforce the possibility that MEQ and MYB cooperate in the maintenance of the transformed state and the tumorigenic potential of these cells.

Sexing of chicken feather follicle, blastodermal and blood cells.

The production of chicken chimeras using donor and acceptor cells which can be of opposite sex has necessitated the utilization of methods developed to distinguish the sex of chickens. We demonstrate one of these methods, based on the polymerase chain reaction which amplifies the EcoRI repeat unit of the fowl W chromosome, and how this technique may be used to sex various cell types in chickens as well as small numbers of blastodermal cells. Our results demonstrate the ability to sex chickens using EcoRI primers, specific for the W chromosome, from as little as 2 ng of female genomic DNA isolated from blood and feathers--the latter being the result of DNA extraction from only one feather. Also evident in this study is the detection of the W chromosome by PCR from approximately 50 blastodermal cells originating from the developing blastodisc at stage X.

Intramagnal insemination of hens can eliminate negative influence of lipofectin on fertilising ability of spermatozoa.

1. After intramagnal insemination egg production decreased drastically during the first two days and was equivalent to egg production of hens inseminated intravaginally for the remaining period of collection. 2. After magnal insemination, the fertility of eggs collected during the first week was 36.2% and only 3.6% during the second week. 3. In the case of intramagnal insemination, egg fertility in the first week was 88.1%, in the second week 81.8% and the third week 52.3%. 4. The eggs laid during the first day after intramagnal insemination were 83.3% fertile, indicating that treated spermatozoa fertilised the newly ovulated egg within 20 minutes of ovulation.

Germline transmission of exogenous genes in chickens using helper-free ecotropic avian leukosis virus-based vectors.

We have used vectors derived from avian leukosis viruses to transduce exogenous genes into early somatic stem cells of chicken embryos. The ecotropic helper cell line, Isolde, was used to generate stocks of NL-B vector carrying the Neo(r) selectable marker and the Escherichia coli lacZ gene. Microinjection of the NL-B vector directly beneath unincubated chicken embryo blastoderms resulted in infection of germline stem cells. One of the 16 male birds hatched (6.25%) from the injected embryos contained vector DNA sequences in its semen. Vector sequences were transmitted to G1 progeny at a frequency of 2.7%. Neo(r) and lacZ genes were transcribed in vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny.

Somatic and germline chicken chimeras obtained from brown and white Leghorns by transfer of early blastodermal cells.

Stage X blastodermal cells were isolated from freshly laid unincubated Brown Leghorn chicken eggs. Five hundred cells from Stage X Brown Leghorn embryos were injected into the subgerminal cavity of White Leghorn unincubated embryos exposed to 550 rad of gamma irradiation from a cesium-137 source. Of 712 White Leghorn embryos that were irradiated and injected with Brown Leghorn blastodermal cells, 52 (7.3%) survived to hatching. Somatic chimerism was examined in the melanocyte population and erythroid lineage. The presence of brown feathers indicating donor cell contribution to melanocyte pigmentation was observed in 23 (44%) out of the 52 hatched chicks. Analysis of blood DNA was performed using a probe that revealed an endogenous retroviral gag fragment specific for the donor genome. Three out of these 23 chimeric chickens exhibited the gag-specific fragment. To test germline chimerism, chickens that reached sexual maturity were mated with Brown Leghorns. Three somatically chimeric hens produced Brown Leghorn progeny at a rate of 30.7, 9.2, and 2.9% respectively, thus proving donor cell contribution to the germline differentiation. Chimeric chickens obtained after injection of nonirradiated embryos exhibited a lower extent of chimerism at the feather level and did not show any chimerism in the erythroid lineage and the germline, thus demonstrating the value of the use of compromised recipient embryos to produce chimeras in chickens. Nevertheless, the extent of somatic chimerism could not be used to predict the germline chimerism.

Analysis of B complex polymorphism in Rous sarcoma progressor and regressor chickens with B-G, B-F, and B-L beta probes.

Molecular polymorphism of the B complex was studied in serologically defined B19 haplotypes by use of class I, class II, and class IV probes in Southern blot experiments in chickens. All chickens studied shared identical class IV restriction patterns. In contrast, class I and class II probes revealed six and five subtypes of B19 haplotype, respectively. These subtypes may be resolved in three homozygous genotypes and their corresponding heterozygous combinations. Previous genetic selection allowed us to distinguish two subpopulations in these B19 chickens with regard to the fate of Rous sarcoma virus (RSV)-induced tumors. Molecular genotyping was applied to B19 chickens challenged with RSV in order to determine whether there is a correlation between one of the molecularly defined subtypes and the progressor/regressor phenotypes of the chickens. None of the molecularly defined subtypes correlated with the progressor or regressor phenotype of the challenged birds.

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes.

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.

Avian retroviral vectors derived from avian defective leukemia virus: role of the translational context of the inserted gene on efficiency of the vectors.

We have constructed retroviral vectors derived from the genome of avian erythroblastosis virus ES4 (AEV ES4). The neo selectable gene was substituted for the original v-erbA or v-erbB oncogenes of AEV, either in the same or in a different reading frames. Recombinant retrovirus were rescued and used to infect chicken embryo fibroblasts or quail QT6 cells. When the neo gene was inserted in the same reading frame as the original oncogene, we obtained (1) a high level of expression of the neo gene, (2) a balanced ration of both genomic and subgenomic RNAs, and (3) high titer recombinant viruses. Conversely, when the neo gene was inserted in a reading frame different from that of the original oncogene, we observed (1) a very low level of expression of the neo protein, (2) a predominance of the viral transcript used as translational template for the neo protein synthesis, and (3) low titer recombinant viruses. One of the vectors was used to transfer a human delta-globin gene into avian cells in culture without detectable rearrangement of this gene, but exhibited a deletion within the conserved noncoding region located between the two original oncogenes. Our data provide information for further construction of double expression vectors. Furthermore, three of the vectors would provide helpful tools to identify genetic elements of the virus genome involved in splicing regulation.

Generation of a helper cell line for packaging avian leukosis virus-based vectors.

We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produced replication-defective virus at high titer but they did not release any replication-competent particles. Proviral DNA was shown to be correctly integrated as well as correctly expressed. Viral RNAs were shown to be correctly translated into gag-related polypeptides.