Molecular cloning and sequencing of a cDNA encoding a beta-thyroid hormone receptor in muscovy duckling.

A cDNA clone encoding a beta-thyroid hormone receptor (TRbeta) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TRbeta sequence showed a high degree of homology. This cDNA was used as a probe to characterize the TRbeta mRNA transcripts expressed in muscovy duckling liver.

In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.

Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.

Epilepsy, antiepileptic drugs, and malformations in children of women with epilepsy: a French prospective cohort study.

We conducted a prospective study of teratogenic effects of antiepileptic drugs (AEDs) in pregnant women with epilepsy in southeast France, comparing malformation rates with those collected by a birth defects registry. We evaluated isolated microcephalies separately. Malformations were seen in 7% of infants of mothers with epilepsy (IME) and in 1.36% of the general population. No significant relationship was found between type and severity of epilepsy and occurrence of malformations or isolated microcephaly. Valproate and phenytoin were the most teratogenic (all malformations). None of the malformations observed in IME whose mothers received valproate, phenytoin, or phenobarbital was seen in IME not exposed to the respective AEDs. Phenytoin plus phenobarbital was more teratogenic than phenobarbital alone. Benzodiazepines, prescribed only in combinations, had a borderline, nonspecific effect on microcephaly.

Evidence for a polymorphism of HLA-G gene.

HLA-G gene polymorphism was analyzed by RFLP using seven restriction enzymes and an HLA-G locus-specific probe. Hybridization of 55 DNAs digested with three enzymes (Taq I, Pst I, and Bgl II) revealed two polymorphic bands in each case. RFLP patterns obtained with Taq I and Pst I corresponded to the same allelic polymorphism and differed from the Bgl II polymorphism. Combining both polymorphisms enabled determination of four alleles. Allelic frequencies were calculated: 40% of the subjects tested had allele 1, 36% had allele 2, 22% had allele 3, and 2% had allele 4. Analyzing the complete HLA class I phenotype revealed strong linkage disequilibrium with the HLA-A locus. The polymorphism described is located in the 3' flanking region of the gene. Moreover, extended HLA-A haplotypes were constructed by combining the HLA-G polymorphism with other class-I-sequence polymorphisms.

Characterization of a cDNA encoding an alpha thyroid hormone receptor in muscovy duckling.

A complementary deoxyribonucleic acid (cDNA) clone encoding an alpha thyroid hormone receptor (TR alpha) from muscovy duckling liver was isolated and sequenced. Comparison with the chicken TR alpha sequence showed a high degree of homology. Despite 45 nucleotide substitutions, the deduced peptide sequence was similar. This cDNA was used as a probe to characterize the TR alpha mRNA transcripts expressed in muscovy duckling liver and skeletal muscle.

[Alcohol and pregnancy]. / Alcool et grossesse.

Alcohol consumption during pregnancy is a major cause of mental retardation in Western countries. Fetal alcohol syndrome (FAS) is mainly characterized by pre- and postnatal stunted growth, neurocognitive disorders, and facial dysmorphism. It compromises the intellectual and behavioral prognosis of the child. Prevention tools exist, through better information of health professionals, for optimal care of high-risk women before, during, and after pregnancy, which would decrease the incidence of SAF in the future.

Influence of the nature of the reporter gene and selection pressure on stability of avian retrovirus vectors.

We have compared the long-term stability of 2 avian non-replicative retroviral vectors in an infected permanent cell line from quail fibroblasts (QT6). Vectors NL53 and NPL, expressing both the neo and LacZ genes under control of cis-acting elements originated from avian erythroblastosis virus (AEV), are similar to each other except for the presence of the phleomycin-resistance SHble gene fused upstream the reporter LacZ gene, in NPL vector. The use of such vectors, with an uniform backbone, to infect QT6 cells, allowed us to demonstrate that stability of the beta-galactosidase activity encoded by the SHble-LacZ fusion gene remains higher than that encoded by the native LacZ gene, as determined in the same conditions of culture. Moreover, stability of the provirus was dependent on the selection pressure. Here we show that stability of beta-galactosidase activity in infected QT6 cells was obtained with high dose selection for the selectable SHble-LacZ fusion gene.

Influence of expression and cis-acting sequences from avian leukosis viruses (ALVs) on stability of (ALV)-based retrovirus vectors.

Defective avian leukosis virus (ALV)-based vectors expressing the neo and LacZ genes were constructed under the control of cis-acting elements originated from 4 avian retroviruses: avian erythroblastosis virus (AEV), Rous associated viruses 1 (RAV-1) and 2 (RAV-2), and the Schmidt Ruppin strain of Rous sarcoma virus subgroup D (SR-RSV-D). We used these vectors to study the long-term stability of beta-galactosidase expression (encoded by the LacZ gene) in a permanent cell line from quail fibroblasts (QT6). Infection of the immortalized QT6 cell line with these vectors resulted in unstable beta-galactosidase expression. We determined whether this instability of provirus expression was correlated with: (1) presence of G418 selection; (2) deletion in the proviral genome; (3) hypermethylation of the proviral genome; (4) position of the neo and LacZ genes in the proviral genome; and (5) the transcriptional activity of the long terminal repeat (LTR) elements of proviral vectors. We observed that G418 selection pressure applied to infected QT6 cells lead to a more stable LacZ gene expression. Moreover, our results suggest a correlation between the stability of proviral gene expression and the level of gene expression driven by the LTR elements and depending on the strain origin of these.

Use of retroviral vectors to introduce and express the beta-galactosidase marker gene in cultured chicken primordial germ cells.

Three methods of isolating primordial germ cells (PGCs) from gonads of 5-day-old chick embryos were compared. PGCs were then cultured in vitro in DMEM/F12 medium containing 10% fetal calf serum. BrdU incorporation showed that at least 10% of the PGC population were dividing, under our culture conditions, during the 2nd day of in vitro culture. During this culture period, PGCs were exposed to avian leukosis sarcoma virus-based retroviral vector pseudotyped with subgroup A envelope, carrying the LacZ reporter gene. X-Gal staining showed that PGCs were permissive to infection, with more than 50% of PGCs expressing the beta-Gal protein. These data represent the first demonstration that PGCs, isolated from gonads of 5-day-old chick embryos, are able to divide in vitro and that it is possible to introduce and express exogenous DNA in chick PGCs maintained in vitro.

Defective retroviral endogenous RNA is efficiently transmitted by infectious particles produced on an avian retroviral vector packaging cell line.

This report describes the contamination of "helper-free" stocks of defective retroviral vector with particles bearing retroviral endogenous RNA. An avian leukosis virus-based packaging cell line was developed from LMH cells that bear the ev1, ev3, and ev6 retroviral endogenous loci. The results show that an endogenous retroviral transcript (ev3) was packaged into virions produced by this packaging cell line and was efficiently transferred along with the vector to target cells. The titer of the ev contaminant particles was estimated at 50-100 CA-p27gag-expressing units/ml of supernatant.

Immune response and resistance to Rous sarcoma virus challenge of chickens immunized with cell-associated glycoproteins provided with a recombinant avian leukosis virus.

The Rous-associated virus 1 env gene, which encodes the envelope gp85 and gp37 glycoproteins, was isolated and inserted in place of the v-erbB oncogene into an avian erythroblastosis virus-based vector, carrying the neo resistance gene substituted for the v-erbA oncogene, to generate the pNEA recombinant vector. A helper-free virus stock of the pNEA vector was produced on an avian transcomplementing cell line and used to infect primary chicken embryo fibroblasts (CEFs) or quail QT6 cells. These infected cells, selected with G418 (CEF/NEA and QT6/NEA, respectively) were found to be resistant to superinfections with subgroup A retroviruses. The CEF/NEA preparations were used as a cell-associated antigen to inoculate adult chickens by the intravenous route compared with direct inoculations of NEA recombinant helper-free virus used as a cell-free antigen. Chickens injected with the cell-associated antigen (CEF/NEA) exhibited an immune response demonstrated by induction of high titers of neutralizing antibodies and were found to be protected against tumor production after Rous sarcoma virus A challenge. Conversely, no immune response and no protection against Rous sarcoma virus A challenge were observed in chickens directly inoculated with cell-free NEA recombinant virus or in sham-inoculated chickens.

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.

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.

Improvement of avian leukosis virus (ALV)-based retrovirus vectors by using different cis-acting sequences from ALVs.

Production and expression of double-expression vectors which transduce both Neo(r) and lacZ genes and are based on the structure of avian leukosis virus were enhanced by using cis-acting sequences (long terminal repeats and noncoding sequences) from Rous-associated virus-1 and Rous-associated virus-2 rather than those of avian erythroblastosis virus previously used in our constructs. Polyclonal producer cells obtained after transfection of these vectors into the Isolde packaging cell line gave rise to titers as high as 3 x 10(5) lacZ CFU/ml, whereas it was possible to isolate clones of producer cells giving rise to titers of more than 10(6) resistance focus-forming units per ml.

Defective RNA packaging is responsible for low transduction efficiency of CAEV-based vectors.

Replication defective retroviral vectors are regularly used for transfer and expression of exogenous genes into dividing cells and in animals. Since lentiviruses are able to infect terminally differentiated and non-dividing cells, their use to produce replication defective vectors may overcome this limitation. We developed two replication-defective lentiviral vectors based on the genome of Caprine Arthritis Encephalitis Virus (CAEV). The first vector (pBNL2) carries the neo and lacZ marker genes. Neo gene is expressed from a genomic RNA and lacZ gene from a subgenomic RNA. The second vector (pCSHL) carries a single fusion gene encoding both phleomycin resistance and beta-galactosidase activity. Replication-competent CAEV was used as helper virus to provide the viral proteins for transcomplementation of these vectors. Our data demonstrated that the genomes of both vectors were packaged into CAEV virions and transduced into goat synovial membrane cells following infection. However, the vector titers remained 3 to 4 logs lower than those of CAEV. Further analysis showed a lack of accumulation of unspliced pBNL2 RNA into the cytoplasm of producer cells resulting in the packaging of pBNL2 sub-genomic RNA only. In contrast, RNA produced from pCSHL vector was correctly transported to the cytoplasm and more efficiently packaged than the pBNL2 sub-genomic RNA as revealed by slot-blot and quantitative RT/PCR analyses. However this higher packaging efficiency of pCSHL genome did not result in a higher transduction efficiency of lacZ gene.