RESUMEN
A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells.
Asunto(s)
Técnicas de Transferencia de Gen , Genes env/genética , Virus de la Leucemia Murina de Moloney/genética , Transducción Genética , Animales , Anticuerpos Monoclonales , Western Blotting , Línea Celular , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Genes gag/genética , Genes pol/genética , Humanos , Fosfatos/farmacología , ARN Mensajero/metabolismo , Ratas , Receptores Virales/metabolismo , Retroviridae/genética , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Hereditary C1q deficiency is a rare disease and up to now only 41 cases have been reported. Since all but 3 cases developed SLE or SLE-like disease, C1q deficiency represents the most powerful disease susceptibility gene identified for the development of SLE in humans. A molecular defect in homozygous C1q deficiency has been identified in 13 families. Four of these families are Turkish in origin and they all share the same mutation which is a CAG to TAG change at codon 186 in the A chain. This led us to investigate whether this mutation might be found in Turkish SLE patients and whether it could cause increased disease susceptibility when expressed in the heterozygous form. METHODS: We screened 65 Turkish lupus patients and 49 healthy Turkish individuals by carrying out an amplification of exon 2 of the A chain and restriction enzyme analysis for the C1qA mutation. RESULTS: We found no other example of this mutation in either the homozygous or heterozygous forms. CONCLUSION: C1q deficiency is one of the very strong disease susceptibility genes in lupus and may cause SLE via a critical role in the physiological clearance of apoptotic cells. However, C1q deficiency caused by a particular mutation in the A chain in a heterozygous form is not found in the Turkish SLE population.
Asunto(s)
Complemento C1q/deficiencia , Complemento C1q/genética , Encuestas Epidemiológicas , Lupus Eritematoso Sistémico/genética , Mutación , Adolescente , Adulto , Alelos , Secuencia de Bases/genética , Niño , Femenino , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , TurquíaRESUMEN
OBJECTIVE: To identify intervals containing systemic lupus erythematosus (SLE) susceptibility alleles in the BXSB strain of mice. METHODS: We analyzed 286 (B10 x [B10 x BXSB]F1) backcross mice for a range of phenotypic traits associated with the development of SLE in BXSB mice. The mice were genotyped using 93 microsatellite markers, and the linkage of these markers to disease was studied by extreme-phenotype and quantitative trait locus analysis. RESULTS: The disease phenotype in these backcross mice was less severe than that in BXSB mice. However, antinuclear antibody production was increased compared with the parental strain. We identified 4 areas of genetic linkage to disease on chromosome 1 (Bxs1-4), 1 on chromosome 3 (Bxs5), and another interval on chromosome 13 which were associated with various aspects of the phenotype. Bxs4 and Bxs5 are located in regions not previously linked to disease in other models of SLE. CONCLUSION: SLE in the BXSB mouse model has a complex genetic basis and involves at least 5 distinct intervals located on chromosomes 1 and 3. There is evidence that different intervals affect particular aspects of the SLE phenotype.
Asunto(s)
Cromosomas/genética , Lupus Eritematoso Sistémico/genética , Alelos , Animales , Anticuerpos Antinucleares/genética , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 13 , Cromosomas Humanos Par 3 , ADN/inmunología , Ligamiento Genético , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Ratones , Ratones EndogámicosRESUMEN
BXSB mice spontaneously develop a lupus-like syndrome that is accelerated by the Yaa gene (Y-linked autoimmune accelerator). We studied the phenotype of disease in (B10 x BXSB)F1 and (BXSB x (B10 x BXSB)F1) backcross mice and genotyped 224 backcross animals to allow a microsatellite-based genome-wide linkage analysis to be conducted. In the backcross population, three intervals on chromosome 1 showed significant linkage to disease, suggesting that multiple loci contribute to the production of autoimmune disease. D1Mit5 at 32.8 cM was linked to development of nephritis (chi(2) = 15.68, p = 7.5 x 10(-5)), as was D1Mit12 at 63.1 cM (chi(2) = 20.17, p = 7.1 x 10(-6)). D1Mit403 at 100 cM was linked to anti-dsDNA Ab production (chi(2) = 17.28, p = 3.2 x 10(-5)). Suggestive linkages to antinuclear Abs and nephritis were identified on chromosome 3, to splenomegaly on chromosome 4, and to anti-ssDNA Ab production on chromosome 10. Chromosome 4 and the telomeric region of chromosome 1 have previously been linked to disease in other mouse models of systemic lupus erythematosus; however, the centromeric regions of chromosome 1 and chromosomes 3 and 10 are unique to BXSB. This implies that, though some loci may be common to a number of mouse models of lupus, different clusters of disease genes confer disease susceptibility in different strains of mice.
Asunto(s)
Mapeo Cromosómico , Marcadores Genéticos/inmunología , Nefritis Lúpica/genética , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/sangre , Cruzamientos Genéticos , Susceptibilidad a Enfermedades , Ligamiento Genético/inmunología , Riñón/patología , Nefritis Lúpica/inmunología , Nefritis Lúpica/mortalidad , Nefritis Lúpica/patología , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Fenotipo , Bazo/patologíaRESUMEN
Two siblings (case 1 and case 2) with homozygous C1q deficiency are described. Both presented with a photosensitive rash, and during follow-up case one developed SLE with nephrotic range proteinuria. Case 2 had microscopic hematuria with a past history of macroscopic hematuria. Renal biopsies revealed mesangioproliferative glomerulonephritis in case 1 and IgA nephropathy in case 2, a new finding in association with C1q deficiency. Since the classical pathway of complement plays a role in the development of antibody responses, the family was also evaluated for the immune response to hepatitis B vaccine. Antibody response to hepatitis B vaccine was normal in both affected members and the rest of the family. The A-, B- and C- chain genes of C1q were amplified by PCR and directly sequenced. A homozygous C to T point mutation was identified in genomic DNA isolated from the patients at codon 186 in the A chain that resulted in a premature stop codon. This mutation was present in both parents and both unaffected sibs in the heterozygous state. This mutation was identical to that previously described in a Slovakian family with C1q deficiency. Because of this finding, a series of 92 genomic DNA samples was screened from ethnically distinct patient groups with SLE to test the hypothesis that this mutation of C1q may be a widespread disease susceptibility gene. No further examples of this mutation were found.
Asunto(s)
Complemento C1q/deficiencia , Complemento C1q/genética , Glomerulonefritis por IGA/genética , Lupus Eritematoso Sistémico/genética , Adolescente , Secuencia de Bases , Niño , Preescolar , Cartilla de ADN/genética , Femenino , Glomerulonefritis por IGA/sangre , Glomerulonefritis por IGA/complicaciones , Glomerulonefritis Membranoproliferativa/sangre , Glomerulonefritis Membranoproliferativa/complicaciones , Glomerulonefritis Membranoproliferativa/genética , Vacunas contra Hepatitis B/inmunología , Homocigoto , Humanos , Inmunización , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/complicaciones , Masculino , Linaje , Mutación Puntual , Reacción en Cadena de la Polimerasa , TurquíaRESUMEN
OBJECTIVE: To describe a new kindred with Clq deficiency and to identify the molecular lesions responsible for complete functional C1q deficiency in this and 2 other previously described kindreds. METHODS: The A-, B-, and C-chain genes of C1q were amplified by polymerase chain reaction, cloned, and sequenced. The DNA sequence was checked for mutations. RESULT: Patient 1 had a homozygous G-to-A change at codon 6 of the C chain, causing an amino acid change from Gly to Arg. Patient 2 had a homozygous deletion of a C nucleotide at codon 43 of the C-chain, causing a frame shift, leading to a premature stop codon at codon 108. Patient 3 had a homozygous C-to-T mutation at amino acid position 41 of the C chain, resulting in a premature stop codon. CONCLUSION: In the homozygous state, the mutations are sufficient to cause complete deficiency of Clq. The mutation in patient 1 has been previously reported in a patient of different ethnic origin. A survey of a series of 158 DNA samples from patients with systemic lupus erythematosus showed no other examples of this mutant allele.