Patent Application: US-45321609-A

Abstract:
the invention relates to various methods of detecting and analyzing protein interactions in a cell , which methods involve the appearance of a specific protein interaction being converted to a permanent detection signal by means of providing , in a manner dependent on said protein interaction , a recombinase activity or protease activity .

Description:
in detail , fig1 to 25 illustrate the following points and embodiments : fig1 depicts the diagrammatic representation of the plasmid construct used for the cre recombinase - dependent two - hybrid system in mammalian cells , with , in detail , the constructs referred to as g5 reporters , g5 - bgal , g5 - egfp and g5 - cre , expressing beta - galactosidase , the enhanced green fluorescent protein and cre recombinase , respectively , under the control of a minimal promoter , the e1b - tata box , and five successive gal4 - dependent enhancer elements from yeast ( upstream activating sequence , uas ); the constructs referred to as g5c reporters , g5c - egfp and g5c - cre , expressing the enhanced green fluorescent protein and cre recombinase , respectively , under the control of the human cmv minimal promoter ( cmvmin ) and five successive gal4 - dependent enhancer elements from yeast ( upstream activating sequence , uas ); the constructs referred to as cmv - stop reporters , cmv - stop / egfp , cmv - stop / bgal and cmv - stop / blasr , being able to express the enhanced green fluorescent protein , beta - galactosidase and the enzyme conferring resistance to blasticidins under the control of a human cmv promoter , if the stop cassette flanked by loxp sequence elements has been removed after cre recombinase activity ; the components of the transcription factor used for the two - hybrid system in mammalian cells being depicted . g refers to the construct for expressing the dna - binding domain of of the yeast gal4 transcription factor ( gal4 dbd ) under the control of the human cmv promoter . v refers to the construct for expressing the transcription activation domain of the herpes simplex virus protein vp16 ( vp16 tad ) under the control of the human cmv promoter . gv refers to the construct for expressing the fusion of the dna binding domain of the yeast gal4 transcription factor ( gal4 dbd ) and the transcription activation domain of the herpes simplex virus protein vp16 ( vp16 tad ) under the control of the human cmv promoter . gx refers to the construct for expressing the dna - binding domain of the yeast gal4 transcription factor ( gal4 dbd ) fused to protein x under the control of the human cmv promoter . vy refers to the construct for expressing the transcription activation domain of herpes simplex virus protein vp16 ( vp16 tad ) fused to protein y under the control of the human cmv promoter . fig2 depicts a flowchart of the cre recombinase - dependent two - hybrid system for detecting constitutive protein - protein interactions . in the case of a specific interaction of gx and vy which is mediated by the protein domains x and y , the result is functional reconstitution of a transcription factor which induces gal4 - dependent expression of the cre recombinase . the activity of the cre protein which is located in the nucleus results in the removal of the transcriptional inactivation element ( stop ) and in permanent activation of downstream reporter genes . fig3 depicts gfp - fluorescence and phase contrast images of the pc12 cell line # 20 after transfection with cmv - stop / egfp and transcription factor gv . the cell line # 20 contains the construct g5c - cre in a stably integrated form and shows no egfp expression after cotransfection with the cre - dependent reporter construct cmv - stop / egfp ( bottom left ). after cotransfection of cmv - stop / egfp with transcription factor gv , a gfp fluorescence can be detected ( top left ). the phase contrast images ( right ) depict a comparable number of cells . fig4 depicts gfp - fluorescence and phase contrast images of the stable pc12 cell line # 20 after transfection with cmv - stop / egfp and gv , three days after neuronal differentiation by ngf . the induction of neuronal differentiation and , connected therewith , prevention of further cell divisions have no influence on the activation of cre recombinase - mediated gfp fluorescence after cotransfection of cmv - stop / egfp with transcription factor gv in the pc12 cell line # 20 ( top left ). the gfp - positive cells depicted under higher magnification show the neuronal morphology ( bottom left ). fig5 depicts gfp - fluorescence and phase contrast images of the stable pc12 cell line # 20 . 4 after transfection with gv . the cell line # 20 . 4 contains the constructs g5c - cre , cmv - stop / egfp and cmv - tkzeo / blasr in a stably integrated form and shows gfp fluorescence only after transfection of transcription factor gv ( top left ). under control conditions , no gfp fluorescence is detectable ( bottom left ). the phase contrast images depict a comparable number of cells ( right ). fig6 depicts gfp - fluorescence and phase contrast images of the stable pc12 cell line # 20 . 4 after transfection with gv and blasticidins selection for four weeks . all cells of the blasticidins - resistant cell clones show a comparable gfp fluorescence , all resistant cell clones are gfp - positive . fig7 depicts gfp - fluorescence and phase contrast images of the stable pc12 cell line # 20 . 4 two days after transfection with gv , g - me2bhlh , v - nd and g - me2bhlh with v - nd . the cell line # 20 . 4 shows gfp fluorescence only after transfection of transcription factor gv ( top left ). after cotransfection of the two - hybrid interaction partners v - nd and g - me2bhlh , no gfp - fluorescence is detectable ( bottom left ). under control conditions , transfection of v - nd or g - me2bhlh , likewise no gfp fluorescence is detectable ( center left ). the phase contrast images depict a comparable number of cells ( right ). fig8 depicts gfp - fluorescence and phase contrast images of the stable pc12 cell line # 20 . 4 two days after transfection with gv , g - me2bhlh , v - nd and g - me2bhlh with v - nd , with addition of tsa . after transfection of transcription factor gv , the cell line # 20 . 4 shows gfp fluorescence ( top left ). after cotransfection of the two - hybrid interaction partners v - nd and g - me2bhlh , gfp fluorescence is likewise detectable under the tsa culturing conditions ( bottom left ). under control conditions , transfection of v - nd or g - me2bhlh , no gfp fluorescence is detectable ( center left ). the phase contrast images depict a comparable number of cells ( right ). fig9 depicts gfp fluorescence and phase contrast images ( small inset image ) of the stable pc12 cell line # 20 . 4 after transfection with gv , g - gbr2cc , v - gbr1cc , g - gbr2cc with v - gbr1cc and g - gbr2ccdel with v - gbr1ccdel . after transfection of transcription factor gv the cell line # 20 . 4 shows gfp fluorescence ( top right ). after cotransfection of the two - hybrid interaction partners g - gbr2cc , v - gbr1cc , gfp fluorescence is likewise detectable ( bottom left ). under control conditions , transfection of an empty vector ( control , top left ) and of the individually transfected interaction partners ( g - gbr2cc or v - gbr1cc , bottom left ), no gfp fluorescence is detectable ( center left ). after cotransfection of plasmids coding for coiled - coiled deletion mutants of the intracellular domains of gbr1 and gbr2 , likewise no gfp fluorescence is detectable ( g - gbr2ccdel and v - gbr1ccdel , bottom inside right ). the phase contrast images depict a comparable number of cells ( small inset figure ). fig1 depicts the quantitative evaluation of facs analysis of the stable pc12 cell line # 20 . 4 two days after transfection with gv , v - gbr1cc , g - gbr2cc and v - gbr1cc together with g - gbr2cc , depicted as the relative number of gfp - positive cells . two days after cotransfection of the interaction partners g - gbr2cc and v - gbr1cc , about 100 times more gfp - positive cells can be detected in comparison with the controls , empty vector ( vector control ) and the individual transfections of g - gbr2cc and v - gbr1cc . fig1 depicts a flowchart of the cre recombinase - dependent two - hybrid system for detecting induced and transient protein - protein interactions . in the case of a stimulus - dependent interaction of gx and vy which is mediated by the protein domains x and y , the result is the functional reconstitution of a transcription factor inducing gal4 - dependent expression of the cre recombinase . the stimulus is represented by the explosion symbol . the stimulation - dependent modification of protein section x is indicated by p . after removal of the cellular modification ( represented by the x above the explosion symbol ), the modification and the protein interaction are removed . the activity of the cre protein which is located in the nucleus results in removal of the transcriptional inactivation element ( stop ) and in permanent activation of downstream reporter genes . the activity of the cre protein may be transient and needs to reach a particular threshold only once . fig1 depicts the results of facs analysis three days after transfection of the stable pc12 cell line # 20 . 4 with a vector control , g - creb , v - cbp / kix and g - creb with v - cbp / kix , with or without forskolin stimulation . the cumulated gfp fluorescence ( top ) and the relative number of gfp - positive cells ( bottom ) without forskolin stimulation ( framed columns ) and after transient forskolin stimulation ( black columns ) are depicted . after cotransfection of the interaction partners , g - creb with v - cbp / kix , the number of detected gfp - positive cells is increased about 12 times after forskolin stimulation . the increase in total fluorescence of the gfp - positive cells is twenty times higher than the unstimulated control . transfection with v - cbp - kix shows virtually no background , while transfection with the g - creb construct has gfp background both in the unstimulated and in the forskolin - stimulated reaction mixture . the likewise strong increase in gfp fluorescence after forskolin stimulation presumably reflects the interaction with endogenously present cbp - like transcriptional cofactors . fig1 depicts the diagrammatic representation of the plasmid constructs used for the cre recombinase - coupled two - switch system in mammalian cells . ss refers to the signal sequence , tag is the extracellular domain encompassing specific epitopes , tm is the pdgf - alpha receptor transmembrane domain . the constructs are described in closer detail : the construct referred to as renin expresses the human renin protease under the control of the human cmv promoter . the construct referred to as tev expresses the nia protease of tobacco etch virus ( tev protease ) under the control of the human cmv promoter . the construct referred to as tm / tev expresses a transmembrane - bound form of the tev protease of the tobacco etch virus under the control of the human cmv promoter . the construct referred to as tm / s / tev expresses a transmembrane - bound form of the tev protease of the tobacco etch virus under the control of the human cmv promoter , said tev protease being separated from the transmembrane domain by a peptide section ( s stands for spacer ). the construct referred to as tm / gv expresses a transmembrane - bound inactive form of the transcription activator gv under the control of the human cmv promoter . the construct referred to as tm / ren / gv expresses a transmembrane - bound inactive form of the transcription activator gv under the control of the human cmv promoter , said transcription activator gv being separated from the transmembrane domain by a peptide section ren . ren represents a human renin protease - specific recognition and cleavage sequence . the construct referred to as tm / tev / gv expresses a transmembrane - bound inactive form of the transcription activator gv under the control of the human cmv promoter , said transcription activator gv being separated from the transmembrane domain by a peptide section tev . tev represents a recognition and cleavage sequence specific for the tev protease . the construct referred to as tm / cre expresses a transmembrane - bound inactive form of cre recombinase under the control of the human cmv promoter . the construct referred to as tm / ren / cre expresses a transmembrane - bound inactive form of cre recombinase under the control of the human cmv promoter , said cre recombinase being separated from the transmembrane domain by a peptide section ren . ren represents a recognition and cleavage sequence specific for the human renin protease . the construct referred to as tm / tev / cre expresses a transmembrane - bound inactive from of cre recombinase under the control of the human cmv promoter , said cre recombinase being separated from the transmembrane domain by a peptide section tev . tev represents a recognition and cleavage sequence specific for the tev protease . the construct referred to as tm / eyfp expresses a transmembrane - bound form of enhanced yellow fluorescent protein ( eyfp ) with c - terminal nuclear localization signals ( nls ) under the control of the human cmv promoter . the construct referred to as tm / ren / eyfp expresses a transmembrane - bound form of enhanced yellow fluorescent protein ( eyfp ) with c - terminal nuclear localization signals ( nls ) under the control of the human cmv promoter , said eyfp - nls protein being separated from the transmembrane domain by a peptide section ren . ren represents a recognition and cleavage sequence specific for the human renin protease . the construct referred to as tm / tev / eyfp expresses a transmembrane - bound form of enhanced yellow fluorescent protein ( eyfp ) with c - terminal nuclear localization signals ( nls ) under the control of the human cmv promoter , said eyfp - nls protein being separated from the transmembrane domain by a peptide section tev . ren represents a recognition and cleavage sequence specific for the human renin protease . fig1 depicts a flowchart of the tev protease - dependent molecular switch for activating a membrane - bound transcription factor and subsequent activation of a reporter or reporter system . coexpression of the tev protease with a transmembrane - bound transcription factor ( gv ) which n - terminally has a tev protease - specific recognition and cleavage site ( tev ) results in the cleavage and subsequent translocalization of transcription factor gv into the nucleus . gv - dependent reporter genes or reporter systems are activated . fig1 depicts the quantitative evaluation of facs analysis two days after transfection of the stable pc12 cell line # 20 . 4 with a control vector ( control ), gv , tm / tev , tevgv and tm / tev together with tev / gv . the relative number of gfp - positive cells in comparison with transfection with the soluble transcription factor gv is shown . the number of detected cells after single transfection with the empty vector , with the transmembrane - bound tev or with the transmembrane - bound gv is below 1 %. after cotransfection of the transmembrane - bound tev with the transmembrane - bound transcription factor gv , the soluble gv can detect on average half of gfp - positive cells , in comparison with transfection under comparative conditions . fig1 depicts the diagrammatic representation of the plasmid constructs used for protein interaction - coupled transcomplementation of fragments of the tev protease in mammalian cells , with , in detail , the construct referred to as ntertev expressing an n - terminal fragment of the tev protease of tobacco etch virus under the control of the human cmv promoter , the construct referred to as ctertev expressing an n - terminal fragment of the tev protease of tobacco etch virus under the control of the human cmv promoter , the construct referred to as ntertev - x expressing an n - terminal fragment of the tev protease of tobacco etch virus as fusion protein with the n terminus of a protein x under the control of the human cmv promoter , the construct referred to as ctertev - y expressing a c - terminal fragment of the tev protease of tobacco etch virus as fusion protein with the n terminus of a protein y under the control of the human cmv promoter , the construct referred to as x - ntertev expressing an n - terminal fragment of the tev protease of the tobacco etch virus as fusion protein with the c terminus of a protein x under the control of the human cmv promoter , the construct referred to as y - ctertev expressing a c - terminal fragment of the tev protease of the tobacco etch virus as fusion protein with the c terminus of a protein y under the control of the human cmv promoter . fig1 depicts a flowchart of the protease - dependent molecular switch for activating a membrane - bound transcription factor after protein interaction - coupled functional reconstitution of tev protease fragments . after coexpression of plasmids which , on the one hand , code for an n - terminal tev fragment - protein x fusion protein and for a c - terminal tev fragment - protein y fusion protein , the proteolytic activity is functionally reconstituted in the case of the specific interaction of the proteins or protein sections x and y . this is followed by a stably expressed or transiently cotransfected membrane - bound transcription factor ( gv ) which contains a tev - specific recognition and cleavage sequence ( tev ) being detached from the membrane . the result is translocalization of gv into the nucleus and activation of a reporter gene or reporter system . fig1 part a depicts the quantitative evaluation of facs analysis two days after transfection of the stable pc12 cell line # 20 . 4 with an expression vector coding for the membrane - bound transcription factor gv containing an n - terminal tev - specific recognition and cleavage sequence ( tm - tev - gv ). the cumulated fluorescence relative to the activity of the membrane - bound tev protease is shown in %. expression vectors were cotransfected with tmtevgv , which code for : control : membrane - bound tev ( tm - tev ), a membrane - bound gbr1cc domain fused to a c - terminal tev protease fragment , from amino acid 71 - 243 ( gbr1cc - ctertev - 71 - 243 ), a membrane - bound gbr2cc domain fused to an n - terminal tev protease fragment , from amino acid 1 - 70 ( gbr2cc - ctertev - 1 - 70 ) and a membrane - bound gbr1cc domain fused to a c - terminal tev protease fragment , from amino acid 71 - 243 ( gbr1cc - ctertev - 71 - 243 ) together with a membrane - bound gbr2cc domain fused to an n - terminal fragment of said tev protease , from amino acid 1 - 70 ( gbr2cc - ctertev - 1 - 70 ). after cotransfection of the tev fragments as fusion proteins of the interaction domains gbr2cc and gbr1cc about 30 % of the activity of the membrane - bound tev protease is obtained . b depicts combinations of further tev fragments fused to the coiled - coil interaction domains of gbr1 and gbr2 . the constructs were cotransfected into pc12 cells with the membrane - anchored tm - tev - gv , a plasmid transcriptionally activatable by gv and coding for cre recombinase , and a reporter plasmid coding for firefly luciferase under the control of a strong promoter . however , transcription of said luciferase was interrupted by a stop cassette located between gene and promoter and flanked by plox . the system was activated by specific transcomplementation of the tev protease , ultimately leading to production of luciferase at the end of the cascade . the bar diagram depicts this activity in rlu ( relative luminescence units ) and compares it with the signal of the intact protease . fig1 depicts the diagrammatic representation of the plasmid constructs used for the protease - coupled endless switch system , with , in detail , the construct referred to as ttevev being an inactive tev protease under the control of the human cmv promoter . insertion of a peptide sequence containing at least one tev - specific recognition and cleavage sequence renders this modified tev protease inactive . after specific proteolytic cleavage by an active tev protease , the modified tev protease ttevev is activated , the construct referred to as eytevfp being an inactive , nonfluorescent enhanced green fluorescent protein ( eyfp ) under the control of the human cmv promoter . insertion of a peptide sequence containing at least one tev specific recognition and cleavage sequence renders this modified eyfp inactive . the modified eytevfp is activated after specific proteolytic cleavage by an active tev protease . fig2 depicts a flowchart of the protease - dependent molecular switch for activating a proteolytically activatable nonfluorescent eyfp variant after protein interaction - coupled functional reconstitution of tev protease fragments . after coexpression of plasmids which , on the one hand , code for an n - terminal tev fragment - protein x fusion protein and for a c - terminal tev fragment - protein y fusion protein , the proteolytic activity is functionally reconstituted in the case of specific interaction of the proteins or protein sections x and y . a stably expressed or transiently cotransfected inactive , proteolytically activatable reporter protein , such as , for example , an appropriately modified eyfp ( ey - tev - fp ), is then specifically proteolytically cleaved and activated . fig2 depicts a flowchart of the protease - dependent molecular endless switch for activating a proteolytically activatable , inactive tev protease and a proteolytically activatable , nonfluorescent gfp variant after protein interaction - coupled functional reconstitution of tev protease fragments . after coexpression of plasmids which , on the one hand , code for an n - terminal tev fragment - protein x fusion protein and for a c - terminal tev fragment - protein y fusion protein , the proteolytic activity is functionally reconstituted in the case of specific interaction of the proteins or protein sections x and y . a stably expressed or transiently cotransfected inactive , proteolytically activatable tev protease ( ttevev ) is then specifically proteolytically cleaved and permanently activated . subsequently , a stably expressed or transiently cotransfected inactive , proteolytically activatable reporter protein such as , for example , an appropriately modified eyfp ( ey - tev - fp ) is specifically proteolytically cleaved and activated . activation of the constitutively expressed proteolytically activatable functional elements results in a molecular endless loop . fig2 depicts the diagrammatic representation of the plasmid constructs used for the protease - coupled reverse switch system , with , in detail , the construct referred to as tevinh expressing a protein inhibitor or peptide inhibitor of the tev protease of tobacco etch virus under the control of the human cmv promoter . the construct referred to as tev - x expressing a fusion protein of the intact tev protease of tobacco etch virus and a protein x under the control of the human cmv promoter . the protein x is fused to the c terminus of the tev protease . the construct referred to as tevinh - y expressing a fusion protein of a tev inhibitor and a protein y under the control of the human cmv promoter . the protein y is fused to the c terminus of the tev inhibitor . the construct referred to as x - tev expressing a fusion protein of the intact tev protease of tobacco etch virus and a protein x under the control of the human cmv promoter . the protein x is fused to the n terminus of the tev protease . the construct referred to as y - tevinh expressing a fusion protein of a tev inhibitor and a protein y under the control of the human cmv promoter . the protein y is fused to the n terminus of the tev inhibitor . fig2 depicts a flowchart of the reverse switch system after induced dissociation of the known interaction of protein x fused to a tev inhibitor and of protein y fused to the intact tev protease , coupled to the two - switch system . the protein x and protein y - mediated interaction of the tev inhibitor with the intact tev protease results in inactivation of the tev protease . after induced removal of the interaction , represented by the explosion symbol , the tev protease is activated . the downstream reporter system depicted here is the two - switch system ( see fig1 ). fig2 depicts the diagrammatic representation of the plasmid constructs used for the protease expression feedback - coupled system for endless activation , with , in detail , the constructs referred to as g5 - tev expressing the tev protease under the control of a minimal promoter , the e1b - tata box , and five successive gal4 - dependent enhancer elements from yeast ( upstream activating sequence , uas ). the constructs referred to as g5c - tev expressing the tev protease under the control of the human cmv minimal promoter , and five successive gal4 - dependent enhancer elements from yeast ( upstream activating sequence , uas ). fig2 depicts a flowchart of the protein interaction - regulated protease expression feedback - coupled system for endless activation . after coexpression of plasmids which , on the one hand , code for an n - terminal tev fragment - protein x fusion protein and for a c - terminal tev fragment - protein y fusion protein , the proteolytic activity is functionally reconstituted in the case of specific interaction of the proteins or protein sections x and y . a stably expressed or transiently cotransfected membrane - bound transcription factor ( gv ) containing a tev - specific recognition and cleavage sequence ( tev ) is then detached from the membrane . the result is a translocalization of gv into the nucleus and activation of two coregulated or independent reporter genes one of which is the intact tev protease . the gv - regulated expressed tev protease results in further cleavage of the constitutively expressed tm - tev - gvs and results in permanent activation of the complete reporter system . the following examples describe the individual embodiments of the method of the invention in more detail . all molecular cloning and transfections were carried out using standard protocols according to sambrook et al ( sambrook - j and russell - d w 2001 ). the functional elements of the plasmid vectors for carrying out the cre recombinase - based reporter system and the application in the two - hybrid system in mammalian cells are diagrammatically depicted in fig1 . construction of the reporter plasmids : the plasmid g5 - cat carries , 5 ′ of the chloramphenicol transferase ( cat ) reporter gene , the tataa box of the human e1b gene and five successive enhancer elements ( upstream activating sequence , uas ) having the optimal recognition sequence for the saccharomyces cerevisiae gal4 transcription factor . the g5 - cat plasmid dna served as starting vector for preparing the g5 reporters used and was cut using combinations of restriction enzymes in such a way that it was possible to remove the cat gene 5 ′ and 3 ′ from the vector backbone and to insert the appropriately prepared reporter gene dna fragments . for this purpose , the cre recombinase of bacteriophage p1 ( cre ) was amplified by the polymerase chain reaction ( pcr ) using specific oligonucleotides and modified 5 ′ by a start codon flanked by the kozak sequence ( kozak 1989 ) ( kozak 1987 ). the plasmid vectors g5c - egfp and g5c - cre were cloned using the plasmids teto - egfp and teto - cre and g5 - cat as backbone . the e1b - tataa box and the cat reporter were removed and replaced with the human cytomegalievirus ( cmv ) minimal promotor and the corresponding reporter gene . coding regions of the dna binding domain ( dbd ) of the yeast transcription factor gal4 and of the transactivation domain ( tad ) of the herpes simplex protein vp16 were amplified by means of pcr from corresponding yeast two - hybrid vectors and cloned into the eukaryotic expression vectors pcmv . the oligonucleotides were designed so as to introduce 5 ′ a restriction cleavage site and a kozak sequence - flanked atg and for the last codon 3 ′ to be in the reading frame with another introduced restriction cleavage site without stop codon . stop codons of the three possible reading frames are located in the vector pcmv 3 ′ of the multiple cloning sequences ( mcs ) so that it was possible to utilize the vectors pcmv - gal4 dbd ( g ) and pcmv - vp16 tad ( v ) ( see fig1 ) as starting vectors for c - terminal fusions with further proteins or protein sections . the vector gv which codes for a fusion protein of gal4 dbd and vp16 tad was prepared starting from the pcmv - gal4 plasmid vector and the pcr product coding for v16 tad , taking into account a continuous reading frame ( see fig1 ). the cre - activatable cmv - stop / reporter constructs were prepared by sequential cloning into pcmv . first , the stop cassettes were generated by pcr , incorporating in each case two loxp sites ( recognition and recombination elements of cre recombinase ) in the same orientation 5 ′ and 3 ′ of a neomycin resistance - and of a zeocin - resistance - conferring element ( neor and tkzeor , see fig1 ). the corresponding reporter gene downstream of cre was then cloned in 3 ′ of the stop cassette . egfp and bgal were introduced 3 ′ of the neor stop cassette and an element conferring resistance to blasticidins was introduced 3 ′ of the tkzeor stop cassette . the functionality of the stop - reporter cassettes was analyzed via transient transfections in cos7 cells . for this purpose , said cassettes were cotransfected together with a control vector or with a cmv - cre plasmid vector , it being possible to observe the activity of the downstream reporters , egfp , bgal and blasr , only after cotransfection with cmv - cre . functional analysis of the components of the cre recombinase - based reporter system via transient transfection into pc12 and cos7 cells the reporter plasmids or combinations of reporter plasmids were tested via transient transfections into the pc12 and cos7 cell lines . for this purpose , between 10 5 and 10 6 pc12 or cos7 cells were electroporated using a genepulser ii with capacity extender module ( biorad , munich , germany ). plasmid dna was purified using the qiafilter method ( qiagen , hilden , germany ). for each electroporation , a total of 5 μg of plasmid dna was always used , filling up with plasmid dna of an empty vector , depending on the reaction mixture . the electroporation was carried out in special cuvettes ( peqlab ) in the appropriate cell growth medium and using the following parameters : cos7 cells , 10 5 cells in 300 μl per reaction mixture , pulses with 250 mv at 500 μf ; pc12 cells , 10 6 cells in 300 μl per reaction mixture , pulses of 220 mv at 960 μf . after electroporation , the cells were transferred to 6 cm or 24 well cell culture dishes and cultured . analysis was carried out usually 12 - 72 h after transfection , depending on the reporter used , using fluorescence microscopy , fac sorting ( egfp ) or by colorimetric detection with x - gal in fixed cells ( bgal ). the average transfection efficiency was about 40 % for cos7 cells and about 30 % for pc12 cells . the amount of dna of the reporter plasmids g5 - bgal , g5 - egfp , g5c - egfp , cmv - stop / egfp and cmv - stop / bgal was always 1 μg per reaction mixture , with the amounts of the cre recombinase reporters , g5 - cre and g5c - cre being varied . the functionality of the reporters was tested via cotransfection with the expression plasmid of the complete transcription activator gv ( 1 μg per reaction mixture ). the results were comparable between pc12 and cos7 cells , with a slightly stronger background but overall higher signal intensity in cos7 cells compared to pc12 cells . the cre - based reporter system used herein is based on the gal4 - dependent transcriptional activation of a cre reporter plasmid . the expressed cre protein can then catalyze the excision of a transcriptional stop cassette flanked by cre recognition and recombination sequences ( loxp sites ) in the same orientation . the activated reporter gene is now under the control of the constitutive human cmv promoter which is very strong in most cell lines , resulting in an enormous increase in the signal . after cotransfection of gv with the g5 - bgal reporter , x - gal staining was detected in a multiplicity of cells after only 12 h . after cotransfection of gv with the g5 - egfp reporter , gfp fluorescence was detected only in cos7 cells in a small number of cells after 72 h . transfections of the g5 - bgal and g5 - egfp reporters exhibited no background activity . after cotransfection of the g5c - egfp reporter plasmid with gv , a markedly higher number of gfp - positive cells , compared to the control transfection without gv , was detected after only 48 h . some gfp - positive cells , however , were also detected in the control transfection . transfection of gv together with cmv - stop / egfp or cmv - stop / bgal exhibited no background . cotransfection of in each case 1 μg of g5 - cre and cmv - stop / egfp showed a very high number of gfp - positive cells after 48 h . the first gfp signals were detected after only 12 h . the best ratio of gv - induced signal to background was obtained by transfection of 50 ng of g5 - cre with in each case 1 μg of cmv - stop / egfp or cmv - stop / bgal and gv . the increased basal promoter activity of the g5c - cre construct made it impossible to reduce the background by reducing the amounts used , as for g5 - cre . the evaluation of the transient transfections for analyzing the components of the cre - based reporter system showed the following : 1 ) the reporter system is characterized by a very high sensitivity . 2 ) due to said high sensitivity , it is not possible to completely remove the background of components of the system in transient transfections . 3 ) using the cre recombinase , it is possible to use egfp as downstream cre reporter with a sensitivity and kinetics comparable to bgal . 4 ) when using a relatively strong basal minimal promoter ( cmvmin ), egfp is also less sensitive as reporter than bgal . application of the cre - recombinase - based reporter system after transient transfection into pc12 and cos7 cells as two - hybrid system for analyzing constitutive protein interactions the application of the cre recombinase - based reporter system in the two - hybrid system in mammalian cells was tested via analysis of known interaction partners ( see fig2 , flowchart of the cre recombinase - dependent two - hybrid system ). most basic helix - loop - helix ( bhlh ) proteins form heterodimeric complexes . the interaction is mediated via two amphipathic helices which form a characteristic four - helix bundle ( ma , rould et al . 1994 ) ( baxevanis and vinson 1993 ). the interaction between the bhlh proteins me2 and nex and , respectively , neurod served as a test system . for this purpose , the bhlh domain of me2 was amplified by pcr and expressed as fusion protein with gal4 - dbd ( g - me2bhlh ). full length nex and neurod were expressed as fusion proteins with vp16 tad ( v - nd and v - nex , respectively ). another known motif which mediates specific protein - protein interactions is the leucine zipper motif in coiled - coil ( cc ) domains ( lupas 1996 ). another test system used were parts of the intracellular sections of gbr1 and gbr2 which in each case contain a cc domain via which they form heterodimers ( kuner , kohr et al . 1999 ). gbr1cc ( l859 - k960 ) and gbr2cc ( i744 - g849 ) were amplified and cloned by means of pcr using specific oligonucleotides . gbr1cc was fused to the c terminus of vp16 ( v - gbr1cc ), and gbr2cc was fused to the c terminus of gal4 ( g - gbr2cc ). deletion mutants of said protein domains , gbr1 ( l859 - k960 δs887 - l921 ) and gbr2 ( i744 - g849δs785 - q816 ) ( v - gbr1ccdel and g - gbr2ccdel ) were used as negative control , and these mutations had previously been shown , by immunoprecipitation and by means of yeast two - hybrid technique , not to interact . an interaction was detected both for the interaction partners g - me2bhlh and v - nex and , respectively , v - nd and for g - gbr2cc and v - gbr1cc in pc12 and cos7 cells , using the cre reporter system and gfp fluorescence as measure . the controls , individual transfections and the coiled - coil deletion constructs ( v - gbr1ccdel and g - gbr2ccdel ), showed no or substantially weaker signals . the following relative strength of interactions was obtained from the experiments : gbr2cc / v - gbr1cc & gt ;& gt ; g - me2bhlh / v - nd & gt ;& gt ; g - me2bhlh / v - nex . the results were confirmed using bgal as two - hybrid reporter . preparation of stable pc12 and cos7 cell lines containing the components of the cre reporter system the results from the experiments of the cre recombinase - based two - hybrid system after transient transfection in mammalian cells indicated that 1 ) the sensitivity of the system was comparable to a beta - galactosidase reporter , even when using an egfp downstream of cre , and 2 ) owing to the increased sensitivity and to the switch mechanism of the cre activity , it was not possible to completely reduce the background . in order to be able to control the background of the system , first a component of the tetracyclin - dependent gene regulation system , which enables expression of the interaction partners to be finally regulated , was stably incorporated into pc12 and cos7 cells ( gossen , freundlieb et al . 1995 ). for this purpose , a dna fragment coding for the tet - dependent transactivator ( tta ) under the control of the cmv promoter was cotransfected with a linearized dna element , neor , which confers resistance to the aminoglycoside g418 . g418 selection ( 400 μg / ml ) was started three days after transfection , and after 3 - 4 weeks cell clones were identified and isolated . the latter were analyzed independently for functional tta expression via cotransfection with a tta - dependent reporter ( teto - egfp ). in each case one pc12 cell clone and one cos7 cell clone with tetracycline - dependent regulation of the gfp reporter were used for the further steps . in the next step , the g5 - cre or g5c - cre dna fragments were cotransfected with a fragment conferring resistance to hygromycinb . four weeks after hygromycinb selection ( 60 μg / ml ), cell clones were isolated and analyzed for gv - dependent cre activity . for this purpose , the cos7 and pc12 cell clones were in each case transfected with cmv - stop / egfp and cotransfected with cmv - stop / egfp and gv and analyzed for gv / cre - dependent induction of gfp fluorescence . all of the cos7 and most of the pc12 cell clones which showed an increase in cre activity due to gv likewise exhibited a certain cre background activity . pc12 cell clone # 20 showed absolutely no constitutive cre expression , i . e . no gfp - positive cell was detectable after transfection with cmv - stop / egfp ( see fig3 , bottom left ). after cotransfection of cmv - stop / egfp and gv on the other hand , a multiplicity of gfp - positive cells were detected ( see fig3 , top left ). pc12 cells are an established cultured cell line of a rat pheochromocytoma and thus originate from sympathoadrenergic tissue of the adrenal medulla ( greene and tischler 1976 ). these cells can be stimulated with nerve growth factor ( ngf ) and differentiate in an ngf - dependent manner to a neuron - related cell type which is postmitotic and forms neuronal processes . in order to test the cre - based reporter system under these postmitotic conditions , gv - transfected pc12 cells of the line # 20 were stimulated with ngf ( 2 . 5 s ngf , 5 ng / ml , promega ) immediately after transfection and analyzed three days after gv - induced gfp - fluorescence ( see fig4 ). the efficiency of the cre reporter system ( lower magnification , fig4 top ) was not influenced by ngf - induced differentiation ( lower magnification , fig4 top ). the neuronal morphology of the cells was not impaired by the cre reporter system ( lower magnification , fig4 top ). in summary , these results demonstrate that the cre reporter system functions in stable pc12 cell clones in a background - free manner , even under postmitotic conditions . in order to stably express all necessary components of the cre - based reporter system , pc12 cells of the line # 20 were cotransfected with the linearized plasmid constructs cmv - stop / egfp ( stop = neor ) and cmv - stop / blasr ( stop = tkzeor ) and selected with zeocin ( 500 μg / ml , invitrogen ). gfp - negative and blasticidins - sensitive cell clones were analyzed via transfection with gv . gfp - positive cells of the cell clone # 20 . 4 were detected two days after gv transfection ( see fig5 ). three to four weeks after gv transfection and blasticidins selection ( 2 μg / ml , calbiochem ), resistant cell clones were identified , and all cells of these clones were gfp - positive ( see fig6 ). in summary , these experiments demonstrate that the cell clone 20 . 4 has stably integrated and functionally expressed all components of the cre - based reporter system . application of the two - hybrid system in the pc12 cell line 20 . 4 for analyzing constitutive protein interactions as described in example 4 , the pc12 cell line 20 . 4 expresses all components of the cre - based reporter system in a completely functional manner . in order to test whether the cell line 20 . 4 can be utilized for application in the two - hybrid system ( see fig2 ), the known interaction partners described in example 3 were analyzed . two days after cotransfection of the interaction partners g - me2bhlh and v - nd , no gfp - positive cells were detectable ( see fig7 , bottom left ). after single transfections of g - me2bhlh and v - nd , gfp - positive cells were likewise not detectable ( see fig7 , central figures , left ). the control , after transfection with gv , showed the expected high number of gfp - positive cells ( see fig7 , top left ). this result , together with the observation of complete absence of constitutive background with respect to uninduced cre expression , suggested that at least the gal4 - dependent cre reporter had been integrated into a region of the heterochromatin and was thus accessible only by very strong transactivators such as gv . therefore , the same two - hybrid analysis was carried out in the following experiments , after transient addition of trichostatin a ( 3 μm , for 12 h , sigma ) ( fig8 ). tsa acts as an inhibitor of deacetylases , and inhibition of deacetylation results in an overall less densely packed , transcriptionally inactive heterochromatin sections . after transient addition of tsa into the culturing medium of pc12 20 . 4 cells , a multiplicity of gfp - positive cells were now detected , two days after transfection of the interaction partners g - me2bhlh and v - nd ( see fig8 , bottom left ). after single transfections of g - me2bhlh and v - nd , no gfp - positive cells were detectable ( see fig8 , central figures , left ). the control , after transfection with gv , showed the expected high number of gfp - positive cells ( see fig8 , top left ). the relatively high concentration of 3 μm tsa for a period of 12 h , however , also resulted in a lower number of surviving cells ( cf . fig7 and fig8 , right - hand column ). the tsa concentration with the best ratio of cell survival and nearly background - free detection of the interaction of g - me2bhlh and v - nd , was 300 μm for 12 h . for the slightly stronger interaction of gbr2cc and v - gbr1cc ( see example 3 ), the experiments were carried out with tsa concentrations of 200 μm tsa for 12 h ( see fig9 ). only after cotransfection of the interaction partners gbr2cc and v - gbr1cc , a multiplicity of gfp - positive cells were detected , after single transfection and cotransfection of the cc deletion mutants , gbr2ccdel and v - gbr1ccdel , no gfp - positive cells were microscopically detectable ( see fig9 , bottom bar ). analysis by fluorescent activated cell sorting ( facs ) confirmed these results ( see fig1 ). in summary , these results demonstrate that it is possible to carry out a two - hybrid analysis in the pc12 cells of the line 20 . 4 and to control the sensitivity and , respectively , the background by addition of tsa . application of the cre recombinase - based reporter system as two - hybrid system for analyzing induced and transient interactions this example describes utilization of the cre recombinase - based reporter system in the two - hybrid approach of detecting a stimulus - induced and transient interaction in vivo . as fig1 diagrammatically shows , transient activation of the cre reporter is sufficient in order to ensure , via the function of the cre recombinase located in the nucleus , permanent activation of downstream reporter . a well - characterized example of an induced protein - protein interaction is the phosphorylation - dependent binding of the transcription activators creb to the transcription coactivator cbp ( creb binding protein ) ( chrivia , kwok et al . 1993 ). for example , protein kinase a ( pka )- mediated phosphorylation of creb at ser133 , in the “ kinase - inducible - domain ( kid )”, results in specific binding to the “ kix ” domain of cbp . pka may be stimulated by adding the adenylate cyclase - stimulating substance forskolin , leading to a transient increase in the intracellular camp level and thus to pka activation . after removing pka stimulation by removing the forskolin from the culture medium of cells , creb - ser133 is rapidly dephosphorylated via active phosphatases endogenous to the cell . for analysis in the two - hybrid system in the pc12 20 . 4 cells , creb was fused c - terminally to gal4 dbd ( g - creb ), and the cbp - kix domain was fused to the c terminus of vp16 tad ( v - cbp - kix ). three days after transfection of pc12 20 . 4 cells with g - creb and v - cbp - kix and corresponding controls , detection of the interaction was analyzed by facs . without forskolin stimulation , neither single nor cotransfection of the constructs resulted in significant activation of the cre / egfp reporter system ( see fig1 , top and bottom graph , framed bars ). after transient forskolin stimulation immediately after transfection ( 4 μm for 12 h ) and facs analysis after 3 days , a distinct increase in the total number of gfp - positive cells and in the cumulated total fluorescence after cotransfection of g - creb and v - cbp - kix were observed ( see fig1 , black bar , right ). a by far smaller but likewise significant increase in the number of gfp - positive cells and total fluorescence was measured in a forskolin - dependent manner for single transfection with g - creb . this can be explained by the likewise stimulated interaction of g - creb with the endogenous cbp or related transcriptional cofactors . the stronger relative increase in total fluorescence with and without stimulation , in comparison with the somewhat smaller relative increase in gfp - positive cells indicates a kinetic component in the system . in summary , these results demonstrate that it is also possible to carry out two - hybrid analyses in the pc12 20 . 4 cells , with interactions which are stimulus - dependent and transient . how transient said interaction is , was shown by chawla and bading . their analyses of creb phosphorylation after short - time calcium signals revealed that s133 phosphorylation results in rapid activation of creb , but the protein is inactive again due to dephosphorylation only after a few minutes ( chawla and bading , 2001 ) ( dephosphorylation of s133 results in dissociation of creb and cbp - kix ). proteolytic activation of a membrane - anchored transcription activator after transient cotransfection and , connected therewith , activation of the reporter system in pc12 20 . 4 membrane anchoring of the gal4 / vp16 fusion protein in pc12 20 . 4 and activation of the reporter system after proteolytic removal . in this example , the feasibility in principle of the protease switch on the membrane in pc12 20 . 4 cells is described . it was the aim to establish a further intracellular mechanism which transduces proteolytic events at the periphery into permanent signals . for this purpose , the gal4 / vp16 fusion protein required for recombinase activation was anchored on the membrane ( tm - gv ) and was then intended to activate via a specific proteolytic cleavage the reporter system in the nucleus . stable localization of gal4 / vp16 on the cell membrane was achieved by fusion to the transmembrane domain of the pdgf ( platelet derived growth factor ) receptor , with insertion and correct orientation of the construct in the membrane being ensured by an n - terminal signal sequence . the efficacy of activation was analyzed via expression of the egfp reporter . for this purpose , the transfected cells were trypsinated after 48 h and the proportion of positive cells was quantitatively determined in an fac analyzer ( facscalibur from bd bioscience ). for this experiment , in each case 1 . 5 × 10 5 pc12 20 . 4 cells were plated on a 24 - well plate and transfected with in each case 0 . 5 μg of the corresponding plasmid dna on the next day ( lipofectamine2000 ; invitrogen ). transient expression of the chimeric membrane protein in pc12 20 . 4 cells resulted in no significant activation of the reporter system ( fig1 ), demonstrating that the gv transcription activator is stably anchored on the membrane . to proteolytically remove the gv transactivator , the bases coding for the 7 amino acid recognition sequence ( enlyfqg ) ( seq id no : 1 ) of the tobacco etch virus ( tev ) nla protease ( tev protease ) were inserted into the dna sequence between pdgf transmembrane segment and gal4 / vp16 . introduction of this or alternative protease cleavage sites did not result in any unspecific release of the gal4 / vp16 fusion protein . coexpression of the tev protease , however , led to efficient cleavage of the tm / tev / gv construct and subsequently to distinct activation of the cre / egfp reporter system . in another step , it was intended to demonstrate that the tev protease is active even after membrane anchoring . the latter is a basic requirement for the interaction analysis of membrane proteins . for this purpose , the tev protease was , analogously to the gal4 / vp16 reporter , n - terminally fused to the transmembrane domain of the pdgf receptor and coexpressed with the tm / tevs / gv construct in pc12 20 . 4 cells . the result showed no significant difference in the activation of the cre / egfp reporter system by soluble and membrane - bound protease . this example underlines the suitability in principle of the method of detecting protein interactions outside the nucleus , in particular on the cell membrane . a precondition for this is the functional coupling of an interaction to the proteolytic cleavage , and this may be carried out by transcomplementation of a protease or , in the case of a low concentration of the partners involved , also by producing a suitable proximity between protease and cleavage site ( fig1 ). analogously to the gal4 / vp16 transcription activator construct , the cre reporter was anchored directly on the cell membrane , with the proteolytic release thereof then leading directly to egfp activation in the nucleus . in the case of weak interactions of very rare proteins , it is possible that the double strategy described is not sensitive enough in order to transduce an interaction on the membrane into a signal . a substantially higher sensitivity is achieved , if , instead of the gal4 / vp16 activator used before , the cre recombinase is coupled directly to the membrane by means of a pdgf transmembrane domain and linked by a protease recognition site . overexpression of the tm - cre construct initially resulted in increased background activity and had to be compensated for by weaker expression . for this purpose , the tm - cre construct was stably transfected into pc12 cells and selected for background - free clones . these cell lines , cotransfected with tev protease and stop - egfp reporter plasmid , subsequently turned rapidly and distinctly green . transcomplementation of the tev protease provides a possibility of converting a protein interaction into the proteolytic removal of a membrane - bound transcription activator . the n1a protease of tobacco etch virus is a member of the family of c4 cysteine peptidases which are structurally homologous to the trypsin - like serine proteases . they have therefore likewise a bilobal β - barrel structure in which three amino acids are characteristically arranged . modeling of the tev protease sequence to a known 3d structure of a related protease ( dengue virus ns3 protease pdb 1bef ) with the aid of the swissmodel software suggests that these amino acids , the “ triads ”, are spread over the two lobes of the structure and are located opposite each other . although the two lobes are physically connected with one another , they seem to fold independently of one another , however . the starting point for transcomplementation was the aim to separate the amino acids of the triads in such a way that they are located on different fragments which could per se form a tertiary structure but which exhibit no activity . the dna for the n - and c - terminal fragments of the tev protease was amplified by means of specific pcr oligonucleotides , introducing 5 ′ an nhei and a 3 ′ kpni restriction cleavage site , and cloned in a plasmid via nhei and kpni to the 3 ′ termini of the “ coiled coil ” domains of gbr1 and gbr2 , which are anchored via the pdgf transmembrane domain ( see examples 3 and 5 ). these constructs were designed in such a way that the interaction of the membrane - anchored “ coiled - coil ” regions recombine the n - and c - terminal fragments of the tev protease to an active form , and this may be detected via removal of the likewise membrane - anchored gal4 / vp16 transactivator in pc12 20 . 4 cells . in order to ensure that none of the two generated tev “ subunits ” has proteolytic activity , they were independently transfected and tested . it was also checked , by cloning the particular gbr deletion mutants ( see example 5 ), that the c - and n - terminal lobes do not gain activity after cotransfection . the experiment revealed that several regions in the tev protease are suitable for transcomplementation , in particular the region between amino acid 60 and 80 and , in particular , the region between 95 and 120 . dividing the protease at these sites led to two inactive fragments , and coexpression of these variants fused to interaction domains in many cases reconstituted the proteolytic activity , with some examples being described in more detail below . the two tev fragments gly1 - 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