A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: application to interaction screening

Abstract

An improved mammalian two-hybrid system designed for interaction trap screening is described in this paper. CV-1/EBNA-1 monkey kidney epithelial cells expressing Epstein-Barr virus nuclear antigen 1 (EBNA-1) were stably transfected with a reporter plasmid for GAL4-dependent expression of the green fluorescent protein (GFP). A resulting clone, GB133, expressed GFP strongly when transfected transiently with transcriptional activators fused to GAL4 DNA-binding domain with minimal background GFP expression. GB133 cells maintained plasmids containing the OriP Epstein-Barr virus replication origin that directs replication of plasmids in mammalian cells in the presence of the EBNA-1 protein. GB133 cells transfected stably with a model bait expressed GFP when further transfected transiently with an expression plasmid for a known positive prey. When the bait-expressing GB133 cells were transfected transiently with an OriP-containing expression plasmid for the positive prey together with excess amounts of empty vector, cells that received the positive prey were readily identified by green fluorescence in cell culture and eventually formed green fluorescent microcolonies, because the prey plasmid was maintained by the EBNA-1/Ori-P system. The green fluorescent microcolonies were harvested directly from the culture dishes under a fluorescence microscope, and total DNA was then prepared. Prey-encoding cDNA was recovered by PCR using primers annealing to the vector sequences flanking the insert-cloning site. This system should be useful in mammalian cells for efficient screening of cDNA libraries by two-hybrid interaction.

Figure 1

Diagram of the GFP-reporter mammalian two-hybrid system with extrachromosomal maintenance of prey expression plasmid. GB133 cells, which express the EBNA-1 protein, harbor a GAL4-dependent GFP reporter plasmid in their chromosomes. For interaction screening, GB133 cells can be stably transfected with an expression plasmid for a GAL4 DNA-binding domain (GAL4DB) fusion protein of bait. Cells can then be transiently transfected with a cDNA library expressing fusion proteins of a transcriptional activating domain (AD) and preys. The two-hybrid interaction will induce transcription of the GFP gene, which is readily detectable by fluorescent microscopy of living cells. Once introduced into GB133 cells by transient transfection protocols, the prey-expressing plasmids harboring the OriP replication origin sequence will be maintained stably because of the presence of the EBNA-1 protein, thus keeping the positive prey-expressing cells permanently green fluorescent. GAL4BE, GAL4 binding element; TATA, TATAA box from adenovirus E1b gene promoter.

Figure 2

GFP expression in GB133 cells. (A) Two-hybrid dependent expression of GFP in GB133 cells. Cells were transiently transfected with expression plasmids for a model bait (GAL4DB-fusion Smad4) and prey (MSG1) together with a β-galactosidase (β-Gal) reporter plasmid. Expression of GFP or β-galactosidase was evaluated 48 h after transfection. Wt + AD, wild-type prey with the transactivating domain; WtΔAD, wild-type prey lacking the transactivating domain; Mut + AD, prey mutant with the transactivating domain that does not interact with the bait. (B) GB133 cells maintain OriP-containing plasmids. Cells were transfected transiently with expression plasmids for GAL4DB or a GAL4DB-fusion CR2 transactivating domain (AD) with or without harboring the OriP sequence. GFP expression was evaluated 7 days after transfection, when cells formed confluent monolayers (phase contrast images are not shown).

Figure 3

Recovery of a model prey (MSG1) plasmid from GB133 cells stably expressing a model bait (GAL4DB-Smad4). (A–C) Cells were transiently transfected with a prey (B and C) or empty vector (A), followed by evaluation of GFP expression by fluorescence microscopy at 48 h after transfection (fluorescent light only in A and B; fluorescent light plus weak white light in C). Arrows indicate strongly green fluorescent cells; arrowhead indicates a weakly green fluorescent cell. (D–I) Maintenance of OriP-harboring plasmid in bait-expressing GB133 cells. Expression plasmids for a GAL4DB-fusion transactivating domain with (E) or without (D and F) harboring the OriP replication origin sequence were transiently transfected into the bait-expressing GB133 cells, and expression of GFP was evaluated at 48 h (D) or at 7 days (E and F) after transfection. (G–I) Phase contrast images of cell monolayers corresponding to fluorescence images of D–F are shown in panels G–I, respectively. (J and K) Detection of prey diluted with an excess of empty vectors. Cells were transiently transfected with an OriP-containing prey expression plasmid together with a 2,000-fold molar excess of empty vector, followed by evaluation of GFP expression at 7 days after transfection. Single prey-transfected cells divided three times after transfection, forming green fluorescent cell clusters, each of which consisted of six to eight GFP-expressing cells (fluorescent light only in J; fluorescent light plus weak white light in K). (L) A green fluorescent microcolony of prey-transfected cells 3 days after subculture of the GFP-expressing cell clusters shown in J (fluorescent light plus weak white light). (M) Recovery of prey cDNA from green fluorescent microcolonies by PCR using primers that annealed to the vector sequences flanking the insert-cloning site. Agarose gel electrophoresis of PCR products is shown. Lane 1, positive control amplification from the prey plasmid; lane 2, negative control amplification from empty vector; lanes 3–8, amplification of prey cDNA from total DNA preparations of five independent GFP-expressing microcolonies.