Effects of long-term fertilization on the diversity of bacterial mercuric reductase gene in a Chinese upland soil

ABSTRACT A potent neutralizing Fab fragment from a long-term survivor of simian immunodeficiency virus (SIVsm) infection was used to construct a recombinant macaque immunoglobulin G1κ (IgG1κ) molecule, designated IgG1-201. A Chinese hamster ovary cell line expressing IgG1-201 was derived by stable transfection and optimized for antibody secretion by methotrexate selection and dihydrofolate reductase gene amplification. IgG1-201 effectively neutralized the homologous, molecularly cloned SIVsmH4 virus but had no activity against the heterologous SIVmac251/BK28 virus. The previously characterized, neutralization-resistant SIVsmE543-3 virus was also not neutralized by IgG1-201. Binding to SIVsmH4 gp120 was enhanced in the presence of recombinant soluble CD4, suggesting that IgG1-201 bound a CD4-induced epitope. IgG1-201 immunoprecipitated the SIVsmH4 but not the SIVsmE543-3 envelope despite a close relationship between these two clones. Immunoprecipitation of a panel of SIVsmH4/SIVsmE543-3 chimeric viruses tentatively assigned the neutralization epitope to the third constant domain, immediately C terminal to the V3 loop. These findings suggest the presence of at least one CD4-induced neutralization epitope on SIV, as is the case with human immunodeficiency virus type 1.

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Improved retroviral transfer of genes into canine hematopoietic progenitor cells kept in long-term marrow culture

Blood ◽

10.1182/blood.v74.1.152.152 ◽

1989 ◽

Vol 74 (1) ◽

pp. 152-155 ◽

Cited By ~ 38

Author(s):

FG Schuening ◽

R Storb ◽

RB Stead ◽

S Goehle ◽

R Nash ◽

...

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

Progenitor Cells ◽

Average Rate ◽

Retroviral Vectors ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Reductase Gene ◽

Successful Transfer

Abstract Amphotropic helper-free retroviral vectors containing either the bacterial neomycin phosphotransferase gene (NEO) or a mutant dihydrofolate reductase gene (DHFR*) were used to infect canine hematopoietic progenitor cells. In previous experiments, successful transfer and expression of both genes in canine CFU-GM were achieved after 24-hour cocultivation with virus-producing cells. The average rate of gene expression was 10% (6% to 16%) as measured by the number of CFU-GM resistant to either the aminoglycoside G418 or methotrexate. In an attempt to increase the efficiency of gene transfer, marrow was cocultured for 24 hours with either NEO or DHFR* virus-producing packaging cells and then kept in long-term marrow culture fed three times with virus-containing supernatant (2 to 5 x 10(6) CFU/mL). After six days, cells were harvested and cultured in CFU-GM assay with and without a selective agent. The average rate of gene expression in CFU- GM in five independent experiments was 46% and ranged from 19% to 87%. In conclusion, the efficiency of gene transfer into canine hematopoietic progenitor cells has been increased fourfold by combining cocultivation with long-term marrow culture as compared with results obtained with cocultivation only.

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