Pierrick Thoraval

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 theNeor selectable marker and theEscherichia 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%.Neor andlacZ genes were transcribedin vitro in chicken embryo fibroblast cultures from transgenic embryos of the G2 progeny.

Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity

The chicken major histocompatibility complex (MHC) genes are organized into two genetically independent clusters which both possess class I and class IIβ genes: the classical B complex and the Restriction fragment pattern-Y (Rfp-Y) complex. In this study, we have examined the role of Rfp-Y genes in transplantation immunity. For this we used three sublines, B19H1, B19H2 and B19H3, derived from a line fixed for B19. Southern blots, PCR-SSCP assays using primers specific for Rfp-Y genes, and Rfp-Y class I allele-specific sequencing show that the polymorphisms observed in B19H1, B19H2 and B19H3 are due to the presence of three different Rfp-Y haplotypes. The Rfp-Y class I (YF) alleles in these three haplotypes are highly polymorphic, and RT-PCR shows that at least two YF loci are expressed in each subline. The three sublines show Rfp-Y-directed alloreactivity in that Rfp-Y-incompatible skin grafts are rejected within 15 days, a rate intermediate between that seen in B-incompatible rejection (7 days) and that observed for grafts within the sublines (20 days). We conclude that Rfp-Y has an intermediate role in allograft rejection, likely to be attributable to polymorphism at the class I loci within this region.

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.

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.