Establishment and characterization of immortalized ovine Sertoli cell lines.

The objective of this study was to generate immortalized Sertoli cell lines from prepubertal lamb testes to facilitate investigations during the course of testicular differentiation. The Sertoli cells were enzymatically isolated and immortalized by transfection, with the sequences coding for the SV40 large T-antigen fused downstream of regulatory elements from the human vimentin gene. The different cell lines were positively stained with antibodies to vimentin and transferrin, in agreement with their Sertoli origin. Reverse transcriptase polymerase chain reaction was used to analyze the specific expression of molecular markers (clusterin/sulfated glycoprotein ISGP-2], follicle-stimulating hormone [rFSH], alpha-inhibin, anti-Müllerian hormone, Wilms' tumor gene [WT-1], steroidogenic factor 1 [SF-1], SRY-related HMG box gene g [SOX9], and sex-determining region of Y chromosome) normally expressed in this cellular type. All were shown to express messenger ribonucleic acids for SGP-2, alpha-inhibin, WT-1, SOX9, and SF-1 (except SF-1 for clone no. 1). Moreover, we performed alkaline phosphatase and receptor tyrosine kinase p145 (c-kit) detection to ensure the absence of contamination by peritubular, germ cells, and Leydig cells. Both tests were negative for all the seven cell lines. These ovine Sertoli cell lines are the first ones obtained from livestock that exhibit specific Sertoli cell characteristics resembling different stages of phenotypic development. They provide useful in vitro model systems for toxicological investigations, coculture, and transfection experiments, making it possible to study signal transduction pathways, cell-cell interactions, and gene expression in species other than rodents.

Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells.

OBJECTIVE: Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated. RESULTS: Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin. CONCLUSIONS: Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.

The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine-RGD peptide containing nuclear localisation properties.

One of the major barriers to efficient gene transfer and expression of nonviral vectors for gene therapy is passage across the nuclear envelope. We have previously shown that an oligolysine-RGD peptide that condenses plasmid DNA and binds to cell surface integrins can mediate increased internalisation of plasmid DNA into cells and synergistic enhancement of gene expression when complexed to a cationic lipid. In this report, we show that this enhancement is due to increased nuclear transfer of the plasmid DNA. We have applied the digitonin-permeabilised cell system that has been well established for the study of the nuclear transport of proteins to examine the nuclear transfer of plasmid DNA. Nuclear transfer of plasmid DNA complexed to an oligolysine-RGD peptide and lipofectamine appears to be an energy-dependent process involving the nuclear pore complex, since it is inhibited at 4 degrees C and by treatment with wheat germ agglutinin or with an antibody to the nuclear pore complex which all block nuclear protein import. In accordance with active nuclear transport, we have shown that all these treatments inhibit expression of a luciferase reporter plasmid in permeabilised cells. Nuclear transfer of pDNA is enhanced in mitotic cells, but cell division is not a prerequisite for transfer. We propose that the oligolysine-RGD peptide acts as a nuclear localisation signal and that the cationic lipid is more important for cell entry and endosome destabilisation than nuclear transfer.

Human immortalized brown adipocytes express functional beta3-adrenoceptor coupled to lipolysis.

Human brown pre-adipocytes were immortalized by microinjection of the genes encoding simian virus 40 T and t antigens under the control of the human vimentin promotor. The transfected pre-adipocytes were cultured for several months with no loss of their morphological characteristics. These cells accumulate lipids and differentiate into adipocytes when treated with insulin, triiodothyronine and dexamethazone. The mRNA of various adipocyte markers was detected by reverse transcriptase-polymerase chain reaction analysis, including hormone-sensitive lipase, lipoprotein lipase, adipsin, glucose transporters 1 and 4, the uncoupling protein (specific of brown adipocytes), and leptin, the product of the ob gene. Pharmacological analyses indicated that the beta3-adrenoceptor is the predominant beta-adrenoceptor subtype in PAZ6 cells and that this receptor subtype is functionally coupled to adenylate cyclase and lipolysis. The immortalization of human adipocytes will permit pharmacological analysis of the human beta3-adrenoceptor function in adipose cells and will allow detailed studies of human adipocyte differentiation.

Characterization of a newly established human bone marrow endothelial cell line: distinct adhesive properties for hematopoietic progenitors compared with human umbilical vein endothelial cells.

Human bone marrow endothelial cells (HBMEC) are intimately involved in the homing of hematopoietic progenitor cells (HPC) to the bone marrow and in the regulation of proliferation and differentiation of these cells. Because availability of primary HBMEC and their capacity to be cultured in vitro are limited, we used isolated HBMEC to establish a cloned cell line by microinjection of a recombinant plasmid expressing simian virus 40 early genes under the control of a deletion mutant of the human vimentin promoter. Serum requirements for growth of a transformed HBMEC line (TrHBMEC) were markedly decreased compared with those of primary cells, and added growth factors were not required for proliferation. Cells took up acetylated low-density lipoprotein normally, bound to Ulex europaeus lectin, and stained positively for von Willebrand factor, P-selectin, CD31, CD34, CD44, very late antigen-5, and intercellular adhesion molecule-2 (ICAM-2). After treatment with TNF-alpha or lipopolysaccharide, TrHBMEC increased surface expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and ICAM-1 in a manner similar to primary HBMEC. In contrast, IL-1 beta elicited much less up-regulation of these adhesion molecules than in primary cells. In previous work, we reported that, in a flow adhesion model, rolling of peripheral blood CD34+ cells on primary HBMEC was E-selectin-dependent, whereas VCAM-1 and ICAM-1 contributed to firm adhesion. In the present study, we show that HPC adhere in a similar way to TrHBMEC. A less-pronounced role for VCAM-1 and ICAM-1 was found in the adhesion of HPC to human umbilical vein endothelial cells. Furthermore, significantly more CD34+ cells adhered to TNF-alpha-stimulated HBMEC and TrHBMEC than to similarly stimulated human umbilical vein endothelial cells. These data emphasize the importance of using microvessel HBMEC for studying the homing of HPC to the bone marrow, and indicate the usefulness of the above-described bone marrow endothelial cell line.

Spatial and temporal patterns of c-kit-expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos.

In the mouse, the Kit receptor and its ligand, the stem cell factor (SCF), are encoded at the W/Kit and Steel loci, respectively. The Kit/SCF transduction pathway is involved in promoting cellular migration, proliferation and/or survival of melanoblasts, hematopoietic progenitors and primordial germ cells. Furthermore, a functional Kit/SCF pathway is required for the development of interstitial cells of Cajal (ICC) in the small intestine. Whereas all c-kit-expressing cells in embryogenesis were not identified, previous studies clearly demonstrated that the c-kit expression pattern extends well beyond cells known to be affected by W mutations. To investigate further Kit function, we specifically marked the c-kit-expressing cells and followed their fate during embryogenesis. A mutation was introduced by gene targeting at the W/Kit locus in mouse embryonic stem cells. The lacZ reporter gene was inserted into the first exon of c-kit, thus creating a null allele, called WlacZ. The lacZ expression reflects normal expression of the c-kit gene in WlacZ/+ embryos. The comparison of the patterns of lacZ-expressing cells between WlacZ/+ and WlacZ/WlacZ embryos allowed us to detect where and when melanoblasts, primordial germ cells and hematopoietic progenitors failed to survive in the absence of Kit. We also observed that ICC express c-kit during embryogenesis. ICC are found identically in WlacZ/+ and WlacZ/WlacZ embryos. Therefore, ICC do not depend on Kit expression during embryogenesis. These results indicate that the function of the c-kit gene is only required for the postnatal development of the ICC. Unexpected sites of c-kit expression were uncovered in embryos, including endothelial, epithelial and endocrine cells. None of these cells are dependent on Kit expression for their migration, proliferation and/or survival during embryogenesis. Nevertheless, we assume that the Kit/SCF pathway could be involved in the growth of transformed endothelial, epithelial and endocrine cells.

Transcription of Y- and X-linked genes in preimplantation ovine embryos.

Because male ovine embryos develop faster than female embryos, the transcription of SRY and ZFY, two genes located on the Y chromosome, was examined in preimplantation stages using the reverse transcriptase polymerase chain reaction (RT-PCR). RNA was extracted from pools of ovine embryos matured and fertilized in vitro then cultured in synthetic oviduct fluid medium and recovered from 24 to 207 hr post-insemination (two-cell up to hatched blastocyst stage). Since primers used to amplify ZFY also amplify the homologue ZFX, located on the X chromosome, transcripts were differentiated by digestion with restriction enzymes. ZFY and ZFX transcripts were present in all stages examined following RT-PCR, whereas transcripts for SRY were undetectable in all investigated stages following either RT nested PCR or Southern analysis. The presence of ZFY transcripts suggests that Y chromosome is transcriptionally active during early ovine preimplantation development. The possible relationship between a faster growth of male embryos and the transcription of Y-linked genes at early stages of development is discussed.

Sex-related differences in the developmental rate of in-vitro matured/in-vitro fertilized ovine embryos.

Sex determination of in-vitro matured/in-vitro fertilized ovine embryos cultured in synthetic oviduct fluid (SOF) medium was performed by the polymerase chain reaction amplification of specific Y DNA sequences so as to test the influence of sex on developmental growth during the preimplantation period. At 144 h post-insemination, embryos with a blastocoel were classified as the fast-developing group, whereas those showing a blastocoel only after this length of time were classified as the slow-developing group. At 144 h post-insemination, fast-developing embryos were cultured separately and some were classified according to the size of their blastocoel. At the end of culture (207 h post-insemination), all embryos were classified according to both their developmental stage and their morphological quality. The male:female sex ratio of fast-developing embryos was significantly higher than the expected ratio of 50%. More males were observed at the most advanced developmental stage at both 144 and 207 h post-insemination. The proportion of males did not differ between the good- and poor-quality groups, although a skewed sex ratio was observed with embryos of better quality at the most developed stage. In conclusion, embryos at the most developed stage were predominantly male and were derived mainly from the fast-developing group, raising the possibility of a deviation in the sex ratio after the transfer of in-vitro matured/in-vitro fertilized ovine embryos.

Influence of culture system and oxygen tension on the development of ovine zygotes matured and fertilized in vitro.

The development and quality of ovine zygotes matured and fertilized in vitro were compared after coculture with oviductal cells (CZB-199 system) and culture in synthetic oviduct fluid medium without cells (SOF system). The effect of two oxygen concentrations (5% and 20%) on the development of ovine zygotes in SOF medium was also studied. More ovine zygotes reached the blastocyst stage when culture in SOF medium was performed in 5% O2 rather than 20% O2. A greater number of blastocysts was obtained after culture in the SOF system than coculture in the CZB-199 system. Proportions of grade I (excellent), II (good), III (fair) and IV (poor) blastocysts did not differ significantly between the SOF and CZB-199 systems. Histological examination of hatched blastocysts revealed a superiority of the SOF system for the following: a greater number of total and trophoblastic cells in grade I and II blastocysts; more endodermic cells in grade I blastocysts, higher mitotic index in the inner cell mass of grade II blastocysts and in total and trophoblastic cells of grade I, II and III blastocysts; more grade III blastocysts with mitosis in the inner cell mass; and a lower pyknotic index in the inner cell mass of grade I, II and III blastocysts. Culture in the SOF system improved the rate and quality of blastocysts in comparison with the CZB-199 system. Furthermore, culture in SOF medium with 5% O2 provided more blastocysts than culture in the presence of 20% O2.

Coculture of ovine zygotes fertilized in vivo or in vitro and positive effect of CZB medium on the development of in vitro fertilized zygotes.

The development and quality of ovine zygotes derived from in vivo (IVOF) or in vitro fertilization (IVF) were compared after coculture on sheep oviductal cells. The same criteria were used to evaluate the coculture of IVF zygotes in CZB medium for 2 d followed by 199 medium for 6 d (CZB-199 coculture) or in 199 medium for 8 d (199 coculture). A higher overall developmental rate to blastocyst stages was obtained with IVOF (65.7%) than with IVF (23.2%) zygotes. More IVF zygotes reached blastocyst stages in CZB-199 (36.7%) than in 199 coculture (22.9%). The morphological aspect did not differ significantly between IVOF and IVF or between 199 and CZB-199 blastocysts. Histological examination revealed no significant difference in the pyknotic and mitotic indices and mean number of cells in the trophoblast and in the inner cell mass of hatched blastocysts between IVOF and IVF or between CZB-199 and 199 cocultures. According to criteria used in this study, the quality of blastocysts was equivalent, independently of fertilization or coculture systems. The use of CZB medium during the first cleavages increases the proportion of blastocysts.

Immortalization of multiple cell types from transgenic mice using a transgene containing the vimentin promoter and a conditional oncogene.

Differentiated clonal cell lines were obtained from transgenic mice carrying a recombinant gene composed of DNA coding for a temperature-sensitive mutant of the simian virus large T antigen under the control of regulatory elements of the human vimentin gene. In response to mitogenic factors the vimentin promoter is activated in the presence of serum in almost all cultured cells independently of their origin. The expression of the T antigen could be controlled both at the level of transcription since the vimentin promoter is growth-regulated and at the level of the protein structure through the temperature stability of the T antigen. Indeed, the switch-off of the oncogene protein is obtained by serum deprivation of the culture and achieved with enhancement of the growth temperature. From transgenic mice several types of clonal differentiated cell lines were established and characterized including melanocytes, macrophages, mesangial, muscle, and endothelial cells. Melanocytes displayed melanin while endothelial cells from brain and heart expressed the related factor VIII and low density lipoprotein absorption capacities. Mesangial cells from kidney exhibited numerous desmosomes. Typical markers of macrophages from bone marrow were observed while skeletal muscle cells fused and contracted.

Flecainide distribution, transplacental passage, and accumulation in the amniotic fluid during the third trimester of pregnancy.

OBJECTIVE: To study the disposition of flecainide acetate and its transplacental passage (both into the fetus and in the amniotic fluid) during the third trimester of pregnancy. DATA SOURCES: Reference articles and books are identified in the text. A literature review is presented. CASE SUMMARY: Flecainide distribution, transplacental passage, and accumulation into the amniotic fluid were studied in a patient at term presenting with a fetal supraventricular tachycardia diagnosed at 33 4/7 weeks of gestation. The fetal tachycardia was accompanied by cardiac failure with placental anasarca and hydramnios. Flecainide 100 mg po bid was prescribed initially; by the time of delivery, the dosage had been decreased to 50 mg bid. At delivery day (39 5/7 weeks), the pharmacokinetics of total flecainide were studied at plateau. DATA SYNTHESIS: The concentrations of flecainide at birth in fetal and maternal blood and in amniotic fluid were 235.4, 241.2, and 6426.5 micrograms/L, respectively. Calculation of a fetomaternal blood accumulation ratio of 0.97 showed that, at this gestational age, flecainide penetrates the placental membrane easily without accumulation in fetal blood. In contrast, the concentration of flecainide in amniotic fluid was approximately 27-fold that measured in maternal peripheral blood. Our results suggest the following: (1) close to term, the metabolic clearance (fetal hepatic clearance) of flecainide offers a high yield and its excretion by the fetal kidney is efficient; (2) given that amniotic fluid is constantly swallowed, it seems that, in contrast to what is seen in adults (relative oral bioavailability > or = 95 percent), the oral bioavailability of flecainide is possibly low in the fetus at term or close to term; under such circumstances, the drug would accumulate passively within the gestational sac; and (3) an alternative explanation is that the concentration in the fetus is, in part, the result of both transplacental crossing of the drug and reabsorption orally from the amniotic fluid. CONCLUSIONS: The regular therapeutic monitoring of flecainide is necessary and sufficient in the mother as the concentrations found appear to accurately reflect the degree of fetal accumulation. Because previous studies in infants and children have indicated few toxic adverse effects attributed to flecainide, it appears that the risk to a sucking infant of ingesting toxic amounts of flecainide in human breast milk is very low. Finally, the child of the patient described here has normal initial growth and development at the present time. The transplacental penetration of a drug can be considered, according to gestational age and the disorder being treated, as being of no consequence, dangerous, or desirable. Flecainide appears to fall into this last category.

Pharmacokinetics and protein binding of ceftriaxone during pregnancy.

The purpose of the present work was to study the pharmacokinetics and the protein binding (free fraction of the drug) of ceftriaxone (CTX) during pregnancy. Nine pregnant women (ages, 20 to 34 years) whose gestational ages ranged from 28 4/7 to 40 5/7 weeks were included. The diagnosis of infection was established in all cases; i.e., four women had chorioamnionitis and five women had pyelonephritis. The following triple antibiotic therapy was infused with the aim of achieving cure: CTX, 2 g once every 24 h (constant rate over 60 min); tobramycin, 3 mg/kg of body weight once every 24 h; and ornidazole, 1 g/day. Two series of blood samples were collected, i.e., during the first day of treatment (on day 1), to establish the primary pharmacokinetic profile of CTX, and at the plateau (on day 7), to evaluate a possible accumulation of the drug. This was an open, noncompartmental study, with each patient serving as her own control. Concentrations of total and unbound CTX in serum were measured by a high-performance liquid chromatographic method. Pharmacokinetic analysis was done by a noncompartmental method. Data were compared by a Wilcoxon t test (a P value of < or = 0.05 was considered significant). Data were also compared with those obtained for healthy subjects who received similar treatments. (i) The tolerance to treatment was excellent, and in all cases patients had a complete remission without premature delivery. (ii) No accumulation of CTX was noted during the treatment, and the profiles of the drug determined at days 1 and 7 were not significantly different.(iii) The pharmacokinetic parameters measured in pregnant patients during the third trimester of pregnancy were similar to those measured in healthy subjects. (iv) Residual concentrations of total and unbound CTX measured at 24 h were greater than the MICs for allegedly susceptible organisms, both on day 1 and at steady state. (v) During the final 3 months of pregnancy, the dosage schedule of CTX (2-g infusion per day) required no particular adjustment (i.e., neither a loading dose nor any increase in the maintenance dose.)

Pharmacokinetics of cyclosporin A during pregnancy; monitoring of treatment and specific assays of cyclosporin, based on five liver transplant patients.

Very little information is available concerning the pharmacokinetic behaviour and monitoring of cyclosporin A (CsA) during pregnancy, notably after liver transplant. Monitoring of blood levels of CsA is considered to be one of the best tools for evaluation of the efficacy of immunosuppressant treatment. The aim of this study was to bring together new information concerning pregnant women receiving immunosuppressant treatment with CsA and, in view of the special pathophysiological status of such patients, to compare pharmacokinetic profiles of changes in blood levels of CsA and of the combination of CsA plus metabolites. Specific (CsA-S kit) and a new non-specific (CsA-NS kit) assays of CsA were carried out in five hospitalized pregnant patients who had received liver transplants between the 6th and the 41st weeks of amenorrhea. The results of five cases investigated lead to the following conclusions: (1) The pharmacokinetic behaviour of native CsA in the pregnant woman between the 6th and 41st weeks of amenorrhea suggests no systemic accumulation nor any radical need for changes in dosage schedule as compared with a non-pregnant patient. (2) Monitoring based upon simultaneous use of the CsA-NS and CsA-S kits may be a source of analytical bias and hence confusion for the physician. (3) Determination of an experimental CsA-NS/CsA-S accumulation ratio (based upon analysis of single concentrations or processing of AUCs) is of interest only if specific assays involve not only CsA itself but also its principal metabolites. (4) Monitoring based upon single measurements of residual CsA levels only, is necessary and adequate. Furthermore, such an approach is less costly.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of microinjected DNA and RNA in early rabbit embryos: changes in permissiveness for expression and transcriptional selectivity.

Gene expression in rabbit early development was investigated by microinjecting LacZ DNA and LacZ RNA in 1-cell and 2-cell embryos. Expression of LacZ DNA could not be obtained before 30-36 hpf, although synthetic LacZ RNA was translated from 12 hpf at the least. The onset of expression of microinjected DNA correlated with the 8- to 16-cell stage. This suggests that before this stage, there is a general negative control of gene expression. The arrest of in vitro development at the 2- to 8-cell stages did not inhibit LacZ expression, which still occurred at 33 hpf. In addition the inhibition of the first cleavage by nocodazole resulted in LacZ expression in 1-cell embryos. Expression of microinjected DNA thus occurs at a fixed time after fertilization and is independent of cleavages and of the second and subsequent DNA replications. Therefore, the changes in permissiveness for the expression of microinjected DNA in rabbit embryos are reminiscent of those in mouse embryos. Transcriptional selectivity in rabbit embryos was compared to that in early mouse embryos. In both species, Sp1-sensitive promoters were active and the promoter of simian virus 40 did not require far upstream enhancers before late cleavage stages; genes driven by the -447, +563 region of murine leukemia virus were repressed. In rabbit, however, the H-2Kb promoter active in mouse was silent. Altogether, the results illustrate a remarkable conservation of the characteristics of the transcription in early rabbit and mouse embryos and the independence of its resumption from the pattern of cleavage.

Transplacental passage of vancomycin during the second trimester of pregnancy.

Pharmacokinetics and drug monitoring of vancomycin were studied at mid-pregnancy in a patient with chorioamnionitis due to Streptococcus agalactiae. The terminal half-life remained in the normal range (4-6 hours) because of an equivalent increase in both volume of distribution and total plasma clearance. Transplacental passage of the drug was observed. Monitoring is mandatory for prolonged vancomycin therapy, and the results should be available within 24 hours. The therapeutic regimen of 15-20 mg/kg every 12 hours was sufficient for this patient's chorioamnionitis. Serum drug levels and renal function should be measured before increasing the vancomycin dosage.

[Determination of the ratio of serum concentrations of tobramycin between fetus and mother at birth. A model of small scale pharmacokinetics]. / Détermination des rapports de concentrations sériques foeto-maternelles de la tobramycine à la naissance. Un modèle de pharmacocinétique réduite.

Thirty women at term presenting with fever greater than or equal to 38 degrees C during labour were given a synergic combination of tobramycin-amoxicillin. Gestational ages ranged from 37 6/7 to 41 2/7 weeks. A single dose of 3 mg/kg of tobramycin was given every day intravenously. Feto-maternal tobramycin concentrations (F/M ratios) were systematically determined at birth and allowed to make a small scale pharmacokinetic study. Two curves were plotted from the pharmacokinetic analysis of maternal and fetal measurements, showing an intravenous and an intramuscular profile respectively. A high positive linear correlation (R = 0.82) was found between the F/M ratios and the time (delta T, -hours-) measured from the last administration to the mother to the time of delivery. Elimination of the aminoglycoside was slowed down in newborns (t1/2 lambda z = 5.1 hours). In newborns tobramycin levels were always less than or equal to 6 mg/l and still greater than or equal to 1 mg/l 6 hours after the last maternal injections. Measurement of F/M ratios allowed to study and compare the fetal and maternal pharmacokinetics facilitating (when necessary) the initial posologic adjustment in newborns. Such small scale pharmacokinetics could be extended, for a given gestational age, to other drugs with narrow therapeutic ranges. However, the size and the homogeneity of the population seem to be most important.

Establishment of porcine cell lines producing a murine recombinant retrovirus in order to transfer the nlslacZ gene into porcine cells.

Swine testis (ST) cell lines producing a murine recombinant retrovirus (RRV) were established in order to transfer the bacterial lacZ gene fused to a nuclear location signal (nlslacZ) into animal cells. ST cells were infected with the supernatant of the cat G355.5LacZ2 cell line which produces amphotropic and xenotropic MMuLVSVnlslacZ-defective RRV and wild amphotropic and xenotropic MMuLV. Expression of the nlslacZ reporter gene was under the transcriptional control of both the SV40 early promoter and the retroviral LTR. ST cells expressing the reporter gene were sorted and cloned by limiting dilutions. Fourteen STLacZ-cell lines were isolated and subsequently tested for virus production. Depending on the host range of the retroviruses, two cell lines (STBF11 and STAA3) produced both a xenotropic recombinant pseudotype and wild retroviruses; another (STAB 10) produced both an amphotropic recombinant pseudotype and wild retroviruses. Southern blot analysis of the producer cell lines was carried out to verify proviral integration. The efficiency of the different pseudotypes in the transfer of the nlslacZ reporter gene to cultured animal cells, including porcine cells, was compared to the pseudotyped RRV produced by cat lines. Our results showed that the xenotropic RRV produced by the porcine STBF 11 cell line has a high titre for cells from different species and led to a higher number of porcine endothelial and lymphoblastoid cells expressing the reporter gene than did RRV produced by the cat packaging cell lines.