A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector.

Abstract

cDNA expression cloning is a powerful method for the rescue and identification of genes that are able to confer a readily identifiable phenotpe on specific cell types. Retroviral vectors provide several advantages over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express genes in a wide range of cell types, including those that form important experimental systems such as the hemopoietic system. We describe here a straightforward and efficient method for generating expression libraries by using a murine retroviral vector. Essentially, the method involves the directional cloning of cDNA into the retroviral vector and the generation of pools of stable ecotropic virus producing cells from this DNA. The cells so derived constitute the library, and the virus they yield is used to infect appropriate target cells for subsequent functional screening. We have demonstrated the feasibility of this procedure by constructing several large retroviral libraries (105 to 106 individual clones) and then using one of these libraries to isolate cDNAs for interleukin-3 and granulocyte-macrophage colony-stimulating factor on the basis of the ability of these factors to confer autonomous growth on the factor-dependent hemopoietic cell line FDC-P1. Moreover, the frequency at which these factor-independent clones were isolated approximated the frequency at which they were represented in the original plasmid library. These results suggest that expression cloning with retroviruses is a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes.The hematopoietic system provides us with an exceptional experimental system for studying the control of cellular proliferation and differentiation. During the past several years our understanding of this complex system has improved considerably, largely because of the isolation of growth factors and their cognate receptors, as well as the genes which encode them. Despite this progress, the manner in which such genes and their products interact to maintain normal hematopoiesis in the bone marrow environment is yet to be clearly elucidated. Although several hematopoietic growth factors are known to be produced by marrow-derived stromal cells in vitro (14,22) (25,27). Moreover, the viral DNA is stably integrated, in a predictable configuration, in the infected cells at one or a few copies per cell. This allows for expansion of individual infected cells displaying a particular phenotype and facilitates recovery of sequences inserted in the provirus. We have tested this system by creating a retroviral cDNA library from activated T cells and isolating the cDNAs for the hematopoietic growth factors interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) by using a functional assay in a factor-dependent cell line.

Molecular Cloning Reveals that the p160 Myb-Binding Protein Is a Novel, Predominantly Nucleolar Protein Which May Play a Role in Transactivation by Myb

“…1B) and used in various combinations to amplify FDC-P1 cDNA by PCR. First-strand cDNA was synthesized from FDC-P1 poly(A) ϩ RNA (4 g) with an oligo(dT 18 ) primer and SuperScript reverse transcriptase (Gibco BRL) as described previously (60). Degenerate oligonucleotides corresponding to peptide 4…”

“…5Ј rapid amplification of DNA ends (RACE) PCR (19) was performed by the method used with the 5Ј-AmpliFINDER kit (Clontech). Firststrand cDNA was synthesized from FDC-P1 poly(A) ϩ RNA (2 g) with a p160 cDNA-derived oligonucleotide (5Ј-TTGTAAAGGCCACCATCATAGTCAAC TGCC-3Ј) and SuperScript reverse transcriptase (Gibco BRL) as described previously (60). 5Ј RACE PCR was undertaken with a p160 gene-specific primer, incorporating a BamHI site (5Ј-CGGATCCGGAAGGTACCCACGTATG-3Ј) and the AmpliFINDER anchor primer (Clontech) under the following conditions: 94°C for 45 s, 60°C for 1 min, and 72°C for 2 min for 30 cycles.…”

Retrovirus-mediated transfer of prothymosin gene inhibits tumor growth and prolongs survival in murine bladder cancer

“…10,26 The coding region of the ProT deletion mutant lacking NLS at its C-terminus was obtained by PCR-based mutagenesis Gene Therapy using plasmid pJ6⍀ProT as the template, and two primers, 5Ј-ACC GAG GAG GAT TAG ACA GGA AAA GGA AAA A and 5Ј-GGT GTC CAC ATC GTC ATC CTC ATC ATC CTC, and subsequently subcloned into pRUFneo.…”

Functional expression cloning reveals proapoptotic role for protein phosphatase 4

“…32 PCR products were sequenced by MWG Biotech. For subcloning, PFU proofreading DNA polymerase was used (Promega), followed by subcloning into pCR-Blunt II-TOPO (Invitrogen).…”