Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes.
In order to identify factors that influence expression by retroviral vectors in hemopoietic cells, we have compared viral RNA levels in cells infected with several different recombinant viruses. All of the vectors tested carry the neomycin resistance gene and provide for the insertion of a second gene which, in these studies, comprised sequences from the myc or myb oncogenes or the gene encoding granulocyte-macrophage colony-stimulating factor. The vectors utilize two different strategies for the coexpression of the two genes: alternate splicing and the use of a separate internal promoter. We found that expression in hemopoietic cells could be increased by substituting sequences from the myeloproliferative sarcoma virus long terminal repeat for those of the Moloney murine leukemia virus long terminal repeat. However, none of the vectors examined was able to express a second gene at levels equivalent to those achieved by the parental vectors carrying only the neomycin resistance gene. The reasons for this varied with the different vectors and included inefficient splicing and/or a reduction in the level of unspliced transcripts upon insertion of a second gene. Although the basis of the latter phenomenon is not clear, it is probably related to the position--near the 5' long terminal repeat--at which the second gene was inserted, since insertion of the same genes near the 3' end of another vector had no effect on viral RNA levels. In an attempt to circumvent some of these problems, we constructed a vector that employs an internal beta-actin promoter. Although this vector could express granulocyte-macrophage colony-stimulating factor sequences in a responsive hemopoietic cell line, the level of granulocyte-macrophage colony-stimulating factor produced was disappointingly low. The results from these studies suggest approaches to the design of improved vectors for effective expression of genes in hemopoietic cells.
Bowtell
D D
DD
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Cory
S
S
Johnson
G R
GR
Gonda
T J
TJ
D000818:Animals; D004351:Drug Resistance; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D005814:Genes, Viral; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D051379:Mice; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D012367:RNA, Viral / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*
D012367:RNA, Viral; D011993:Recombinant Fusion Proteins