Solid-phase differential display and bacterial expression systems in selection and functional analysis of cDNAs.
Differential gene expression can be expected during activation and differentiation of cells as well as during pathological conditions, such as cancer. A number of strategies have been described to identify and understand isolated differentially expressed genes. The differential display methodology has rapidly become a widely used technique to identify differentially expressed mRNAs. In this chapter we described a variant of the differential display method based on solid-phase technology. The solid-phase procedure offers an attractive alternative to solution-based differential display because minute amounts of sample can be analyzed in considerably less time than previously. The employed solid support, monodisperse super paramagnetic beads, which circumvents precipitation and centrifugations steps, has also allowed for optimization of the critical enzymatic and preparative steps in the differential display methodology. We also described how bacterial expression can be used as a means to elucidate gene function. An efficient dual-expression system was presented, together with a basic concept describing how parallel expression of selected portions of cDNAs can be used for production of cDNA-encoded proteins as parts of affinity-tagged fusion proteins. The fusion proteins are suitable both for the generation of antibodies reactive to the target cDNA-encoded protein and for the subsequent affinity enrichment of such antibodies. Affinity-enriched antibodies have proved to be valuable tools in various assays, including immunoblotting and immunocytochemical staining, and can thus be used to localize the target cDNA-encoded protein to certain cells in a tissue section or even to a specific cell compartment or organelle within a cell. High-resolution localization of a cDNA-encoded protein would provide valuable information toward the understanding of protein function.
Ståhl
S
S
Department of Biochemistry and Biotechnology, KTH, Royal Institute of Technology, Stockholm, Sweden.
Odeberg
J
J
Larsson
M
M
Røsok
O
O
Ree
A H
AH
Lundeberg
J
J
D000818:Animals; D000906:Antibodies; D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis* / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D008207:Lymphatic Metastasis; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D012709:Serum Albumin / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic
D000906:Antibodies; D017931:DNA Primers; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin