the first nonviral and episomally replicating vector , pepi , was constructed in the late 1990s and is based solely on chromosomal elements .
the functional element of this vector type is an expression unit linked to a chromosomal scaffold / matrix attached region ( s / mar ) .
deletion of this transcription unit or insertion of a transcription termination site upstream of the s / mar results in integration or vector loss , suggesting that an ongoing transcription into the s / mar is essential for its episomal replication and maintenance .
the s / mar - based pepi vector replicates autonomously with low copy number in all cells tested , including primary cells , was successfully used for the efficient generation of genetically - modified pigs , and is mitotically stable in the absence of selection over hundreds of generations .
variations of the pepi vector harboring a human -globin gene transcription unit including the locus control region was fully functional in erythroid cells demonstrating its gene therapy potential . in stably established cell clones
, pepi replicates once per cell cycle during early s - phase and associates with early replicating foci .
3060% of transfected cells show detectable expression of the transgene egfp , although the vector is retained as an episome in all cells .
moreover , the frequency of which the vector establishes in transfected cells is slow : about 95% of all vector molecules that reach the nucleus after transfection are lost during the following cell divisions .
it is assumed that vector establishment is a stochastic event and depends on the nuclear compartment the vector reaches , similar to all other eukaryotic autonomous replicons . since nonviral
s / mar containing vectors represent an exciting alternative to virus - based vectors , considerable efforts have been undertaken for their improvement ( reviewed in ref . ) .
here we focus on chromosomal , cis - acting elements that may be capable to improve efficiency and transgene expression of s / mar - based vectors .
we introduced a novel class of genetic element into pepi - egfp recently designated as a ubiquitous chromatin - opening elements ( ucoe ) .
these elements are responsible for establishing a transcriptionally competent open chromatin structure at regions of the genome that consist only of ubiquitously expressed housekeeping genes .
the ucoe spanning the dual , divergently transcribed promoters of the human tbp - psmb1 and hnrpa2b1-cbx3 housekeeping genes was shown to mediate stable expression of transgenes even when integrated in heterochromatic regions and reduce variegation effects . in combination with the immediate early promoter / enhancer of the human cytomegalovirus ( hcmv ) ,
the ucoe derived from the hnrpa2b1-cbx3 locus ( a2ucoe ) mediates improved levels of expression and proportion of cells expressing the transgene at detectable levels .
another cis - acting element we examined is the 5-hs4 chicken--globin insulator ( chs4 ) , which has been considered as an ideal candidate for use in gene transfer applications due to its barrier and enhancer - blocking function .
two initial studies employed the chs4 1.2  kb insulator sequence in gammaretroviral vectors flanking the transgene and demonstrated a significantly increased fraction of cells expressing the respective transgenes . following these initial reports , a number of studies have been conducted using retroviral vectors flanked with either the whole or parts of the 1.2  kb chs4 insulator , including gammaretroviral vectors and lentiviral vectors .
overall , it was demonstrated that inclusion of the 1.2  kb chs4 fragment increased the probability and/or stability of vector transgene expression and reduced position - effect variegation . besides border and enhancer - blocking functions ,
there is also evidence that suggests insulators are involved in chromatin organization and can anchor active domains to the nuclear periphery , close to the nuclear pore . within this study both elements , a2ucoe and chs4 , were inserted into the prototype pepi - egfp , and plasmid behavior ( episomal replication , establishment efficiency , long - term maintenance , and transgene expression ) was examined .
we demonstrate here that both elements have a remarkable impact on transgene expression and establishment efficiency of s / mar based vectors , without interfering with the episomal behavior of s / mar vectors ; and may therefore be suitable for further studies with gene therapeutic focus .
due to its low establishment efficiency and transgene expression , the cis - acting elements a2ucoe and chs4 were introduced into the episomal vector pepi - egfp ( figure 1a ) in an effort to improve both establishment efficiency ( number of cells in which pepi is stably maintained ) and transgene expression .
the a2ucoe element was inserted upstream of the cmv promoter resulting in pepi - ucoe ( figure 1b ) , while the chs4 element was inserted downstream and in opposite direction of the s / mar element , resulting in pepi - hs4opp ( figure 1c ) .
episomal maintenance of these pepi derivates was ascertained by southern blot analysis using dna from hirt extracts digested with a restriction enzyme that cuts the plasmid only once and either mboi or dpni , respectively .
complete digestion with mboi , which is inhibited by dam dna methylation , indicates cellular replication and was detected for all constructs ( figure 1d ) . in no case , we observed hybridization to chromosomal dna .
cho cells were transfected with pepi - egfp , pepi - ucoe , and pepi - hs4opp and plasmid establishment was determined using a colony - forming assay as previously described .
establishment of the original pepi - egfp was compared with its derivates , pepi - ucoe and pepi - hs4opp ( figure 2a ) .
the original pepi - egfp vector and pepi - ucoe established episomally with a similar efficiency ( pepi - egfp : 9.5    7.25% ; pepi - ucoe : 11.2    9.04% ) .
however , for pepi - hs4opp establishment efficiency was significantly increased ( 25.3    13.98% ; p = 0.028 ) .
after an initial selection period ( 14 days , 400 g / ml g418 ) , cells were cultured in the absence of the antibiotic and transgene expression ( number of egfp expressing cells and mean fluorescent intensity ( fi ) ) was determined in flow cytometry analysis over a time period of at least 60 days . in a mixed population , all pepi plasmids were stably maintained in the absence of drug selective pressure ( figure 2b , c ) .
in addition , a mixed pepi - ucoe population exhibited not only an increased fraction of egfp - expressing cells ( figure 2b ; day 20 : pepi - egfp 9.5    4.8% , pepi - ucoe 53.2    21.2% , p = 0.025 ; day 60 : pepi - egfp 13.7    10.5% , pepi - ucoe 53%  23.2 , p = 0.052 ) , but also a significantly increased expression level ( fi ) when compared with pepi - egfp and pepi - hs4opp ( figure 2c ; day 20 : pepi - egfp 305.5    2.7 , pepi - ucoe 684    68.6 , p = 0.016 ; day 60 : pepi - egfp 283    75 , pepi - ucoe 583.5    83 , p = 0.064 ) .
we next generated and analyzed single - cell clones cho / pepi - egfp#14 , cho / pepi - ucoe#3 , and cho / pepi - hs4opp#1 as described previously . during generation of single - cell clones
, we observed a high variation in percentage of egfp - expressing cells and mean fi in different cho clones for pepi - egfp ( 0.2796% of expressing cells with fi 96905 ) and pepi - hs4opp ( 097% of expressing cells with fi 0788 ) .
in contrast , clones containing pepi - ucoe displayed a more homogenous pattern of egfp - expressing cells ( 1856% ) and expression level ( fi 201848 ) .
of each construct , we chose a clone showing the best combination of high percentage of expressing cells and expression level , respectively .
the number of plasmid copies per cell for each clone was determined using quantitative pcr and specific primers within the pepi - vectors ( figure 3a ) . the original pepi - egfp plasmid and its derivative pepi - hs4opp established in cho cells with five to ten copies / cell ( cho / pepi - egfp#14 : 5.85    3.1 ; cho / pepi - hs4opp#1 : 4.54    0.07 ) , while cho / pepi - ucoe#3 established at a slightly higher copy number ( cho / pepi - ucoe#3 : 12.3    2.6 ) .
long - term maintenance of each single clone was analyzed at regular periodic intervals using flow cytometry .
clone cho / pepi - egfp#14 showed 89.9% , cho / pepi - ucoe#3 30.5% , and cho / pepi - hs4opp#1 97.5% egfp - expressing cells ( figure 3b ) .
again , clone cho / pepi - ucoe#3 showed a significantly higher level of expression compared with cho / pepi - egfp#14 and cho / pepi - hs4opp#1 ( cho / pepi - egfp#14 fi 145 ; cho / pepi - ucoe#3 fi 527 ; cho / pepi - hs4opp#1 fi 76 ) .
normalization of the fi to episome copy numbers resulted in a twofold increased fi for pepi - ucoe over pepi - egfp , and 2.5-fold increased fi for pepi - ucoe over pepi - hs4opp . concerning the high expression level of pepi - ucoe , it has been shown recently that the a2ucoe is marked with histone modifications associated with actively transcribed chromatin . therefore , we examined the histone h3 acetylation status across the expression cassette of pepi - ucoe and its corresponding sequences in pepi - egfp using cho / pepi - ucoe#3 and cho / pepi - egfp#14 , respectively ( figure 4 ) .
the level of histone h3 and acetylated h3k9/14 ( h3k9/14ac ) detected in pepi - egfp were set to 1 and used as a reference against which could be compared the values obtained for pepi - ucoe .
surprisingly , in cho / pepi - ucoe#3 we found a distinctly reduced level of h3k9/14ac in the promoter region ( 3cmv , 0.51-fold ) , spanning the transcription start site of egfp ( tss , 0.25-fold ) , and in the egfp gene ( 5egfp , 0.29-fold ; 3egfp , 0.38-fold ) compared with pepi - egfp .
remarkably , these reduced h3k9/14ac levels were accompanied with a reduced level of overall histone h3 ( 3cmv , 0.47-fold ; tss , 0.53-fold ; 5egfp , 0.55-fold ; 3egfp , 0.62-fold ) , indicating an open chromatin structure accompanied with a lower number of nucleosomes . however , looking in the 3 region of the a2ucoe ( upstream the cmv promoter ) we found an elevated overall level of h3 ( 2.8-fold ) and h3k9/14ac ( 1.7-fold ) , compared with the corresponding sequence in pepi - egfp . since insulator sequences are thought to be involved in chromatin organization , we suggest that the increased establishment efficiency of pepi - hs4opp may be due to an interaction of chs4 with the nuclear matrix . to this hypothesis we performed a nuclear fractionation procedure with the stably established clones cho / pepi - egfp#14 and cho / pepi - hs4opp#1 , to determine
if the episomal vectors they contain are associated with the nuclear matrix .. in these experiments , chromatin was digested with five restriction enzymes that do not cut in pepi - egfp and pepi - hs4opp , together with either ecori ( linearizes both plasmids ) , xmii ( releasing s / mar ) , or xmai ( releasing chs4 ) .
after nuclear fractionation , samples were treated with proteinase k and purified dna was subjected to quantitative pcr to trace the complete vectors as well as their s / mar and chs4 depleted variants in the nuclear matrix fraction .
the matrix fraction contains matrix proteins and the core filament network . in its complete version ( ecori linearized , with s / mar ) , pepi - egfp resides in the matrix fraction ; the detected amount of linearized vectors in the matrix fraction was set to 1 and relative vector amounts of the depleted variants are plotted in figure 5 . upon depletion of the s /
mar with xmii the amount of pepi - egfp vector dna in the matrix fraction is decreased by 16% ( 0.84    0.1-fold over linearized pepi - egfp figure 5a ) . in contrast , the amount of pepi - hs4opp remained nearly unchanged upon depletion of the s / mar ( 0.96    0.13-fold over linearized pepi - hs4opp ) . to test whether the chs4 element is involved in the association with the nuclear matrix
, we released the chs4 element using xmai ; pepi - hs4opp vector dna was again traced in the matrix fraction . upon depletion of the s /
mar element the amount of pepi - hs4opp vector dna in the matrix fraction remained nearly unchanged ( 0.83-fold over linearized pepi - hs4opp ) , while there was a marked reduction of 35% of pepi - hs4opp dna after depletion of the chs4 element ( 0.65-fold over linearized pepi - hs4opp figure 5b ) .
using a similar approach , endogenous dna in nuclear matrix preparations was measured by preserving native attachment sites using cho / pepi - hs4opp#1 cells .
proteins were stripped from nuclei with a mild detergent and subsequently dna was digested with dnase i. remaining dna fragments were precipitated and used for quantitative pcr analyses with total dna extracted from cho / pepi - hs4opp#1 serving as reference and set arbitrarily to 1 . here ,
the s / mar sequence in pepi - hs4opp could be amplified , while the cmv promoter sequence was depleted by the digestion ( data not shown ; 3cmv 0.071    0.05 , 3s / mar 0.302    0.19 , p = 0.055 ) . within the 1.2  kb chs4 element , the 3hs4 fragment could be amplified , while the 5hs4 fragment was amplified at very low levels and apparently reduced by digestion ( data not shown ; 3hs4 0.29 
a protein that mediates many insulator functions and is involved in a broad range of chromatin regulatory events is the ccctc - binding factor ( ctcf ) .
using chromatin immunoprecipitation ( chip ) , we examined binding of ctcf to pepi - hs4opp plasmid dna .
we detected a low signal for the 3cmv and 3s / mar region at background levels ( 1.85    2.5% of input dna ) , and a slightly increased signal for the 3hs4 region ( 2.5    0.44% of input dna ) .
for the 5hs4 region a distinct signal for ctcf binding was detectable ( 11.5    9% of input dna ; figure 6 ) .
due to its low establishment efficiency and transgene expression , the cis - acting elements a2ucoe and chs4 were introduced into the episomal vector pepi - egfp ( figure 1a ) in an effort to improve both establishment efficiency ( number of cells in which pepi is stably maintained ) and transgene expression .
the a2ucoe element was inserted upstream of the cmv promoter resulting in pepi - ucoe ( figure 1b ) , while the chs4 element was inserted downstream and in opposite direction of the s / mar element , resulting in pepi - hs4opp ( figure 1c ) .
episomal maintenance of these pepi derivates was ascertained by southern blot analysis using dna from hirt extracts digested with a restriction enzyme that cuts the plasmid only once and either mboi or dpni , respectively .
complete digestion with mboi , which is inhibited by dam dna methylation , indicates cellular replication and was detected for all constructs ( figure 1d ) . in no case , we observed hybridization to chromosomal dna .
cho cells were transfected with pepi - egfp , pepi - ucoe , and pepi - hs4opp and plasmid establishment was determined using a colony - forming assay as previously described .
establishment of the original pepi - egfp was compared with its derivates , pepi - ucoe and pepi - hs4opp ( figure 2a ) .
the original pepi - egfp vector and pepi - ucoe established episomally with a similar efficiency ( pepi - egfp : 9.5    7.25% ; pepi - ucoe : 11.2    9.04% ) .
however , for pepi - hs4opp establishment efficiency was significantly increased ( 25.3    13.98% ; p = 0.028 ) .
after an initial selection period ( 14 days , 400 g / ml g418 ) , cells were cultured in the absence of the antibiotic and transgene expression ( number of egfp expressing cells and mean fluorescent intensity ( fi ) ) was determined in flow cytometry analysis over a time period of at least 60 days . in a mixed population , all pepi plasmids were stably maintained in the absence of drug selective pressure ( figure 2b , c ) .
in addition , a mixed pepi - ucoe population exhibited not only an increased fraction of egfp - expressing cells ( figure 2b ; day 20 : pepi - egfp 9.5    4.8% , pepi - ucoe 53.2    21.2% , p = 0.025 ; day 60 : pepi - egfp 13.7    10.5% , pepi - ucoe 53%  23.2 , p = 0.052 ) , but also a significantly increased expression level ( fi ) when compared with pepi - egfp and pepi - hs4opp ( figure 2c ; day 20 :
pepi - egfp 305.5    2.7 , pepi - ucoe 684    68.6 , p = 0.016 ; day 60 : pepi - egfp 283    75 , pepi - ucoe 583.5    83 , p = 0.064 ) .
we next generated and analyzed single - cell clones cho / pepi - egfp#14 , cho / pepi - ucoe#3 , and cho / pepi - hs4opp#1 as described previously . during generation of single - cell clones , we observed a high variation in percentage of egfp - expressing cells and mean fi in different cho clones for pepi - egfp ( 0.2796% of expressing cells with fi 96905 ) and pepi - hs4opp ( 097% of expressing cells with fi 0788 ) .
in contrast , clones containing pepi - ucoe displayed a more homogenous pattern of egfp - expressing cells ( 1856% ) and expression level ( fi 201848 ) .
of each construct , we chose a clone showing the best combination of high percentage of expressing cells and expression level , respectively .
the number of plasmid copies per cell for each clone was determined using quantitative pcr and specific primers within the pepi - vectors ( figure 3a ) . the original pepi - egfp plasmid and its derivative pepi - hs4opp established in cho cells with five to ten copies / cell ( cho / pepi - egfp#14 : 5.85    3.1 ; cho / pepi - hs4opp#1 : 4.54    0.07 ) , while cho / pepi - ucoe#3 established at a slightly higher copy number ( cho / pepi - ucoe#3 : 12.3    2.6 ) .
long - term maintenance of each single clone was analyzed at regular periodic intervals using flow cytometry .
clone cho / pepi - egfp#14 showed 89.9% , cho / pepi - ucoe#3 30.5% , and cho / pepi - hs4opp#1 97.5% egfp - expressing cells ( figure 3b ) .
again , clone cho / pepi - ucoe#3 showed a significantly higher level of expression compared with cho / pepi - egfp#14 and cho / pepi - hs4opp#1 ( cho / pepi - egfp#14 fi 145 ; cho / pepi - ucoe#3 fi 527 ; cho / pepi - hs4opp#1 fi 76 ) .
normalization of the fi to episome copy numbers resulted in a twofold increased fi for pepi - ucoe over pepi - egfp , and 2.5-fold increased fi for pepi - ucoe over pepi - hs4opp .
concerning the high expression level of pepi - ucoe , it has been shown recently that the a2ucoe is marked with histone modifications associated with actively transcribed chromatin . therefore , we examined the histone h3 acetylation status across the expression cassette of pepi - ucoe and its corresponding sequences in pepi - egfp using cho / pepi - ucoe#3 and cho / pepi - egfp#14 , respectively ( figure 4 ) .
the level of histone h3 and acetylated h3k9/14 ( h3k9/14ac ) detected in pepi - egfp were set to 1 and used as a reference against which could be compared the values obtained for pepi - ucoe .
surprisingly , in cho / pepi - ucoe#3 we found a distinctly reduced level of h3k9/14ac in the promoter region ( 3cmv , 0.51-fold ) , spanning the transcription start site of egfp ( tss , 0.25-fold ) , and in the egfp gene ( 5egfp , 0.29-fold ; 3egfp , 0.38-fold ) compared with pepi - egfp .
remarkably , these reduced h3k9/14ac levels were accompanied with a reduced level of overall histone h3 ( 3cmv , 0.47-fold ; tss , 0.53-fold ; 5egfp , 0.55-fold ; 3egfp , 0.62-fold ) , indicating an open chromatin structure accompanied with a lower number of nucleosomes . however , looking in the 3 region of the a2ucoe ( upstream the cmv promoter ) we found an elevated overall level of h3 ( 2.8-fold ) and h3k9/14ac ( 1.7-fold ) , compared with the corresponding sequence in pepi - egfp .
since insulator sequences are thought to be involved in chromatin organization , we suggest that the increased establishment efficiency of pepi - hs4opp may be due to an interaction of chs4 with the nuclear matrix . to this hypothesis we performed a nuclear fractionation procedure with the stably established clones cho / pepi - egfp#14 and cho / pepi - hs4opp#1 , to determine
if the episomal vectors they contain are associated with the nuclear matrix .. in these experiments , chromatin was digested with five restriction enzymes that do not cut in pepi - egfp and pepi - hs4opp , together with either ecori ( linearizes both plasmids ) , xmii ( releasing s / mar ) , or xmai ( releasing chs4 ) .
after nuclear fractionation , samples were treated with proteinase k and purified dna was subjected to quantitative pcr to trace the complete vectors as well as their s / mar and chs4 depleted variants in the nuclear matrix fraction .
the matrix fraction contains matrix proteins and the core filament network . in its complete version (
ecori linearized , with s / mar ) , pepi - egfp resides in the matrix fraction ; the detected amount of linearized vectors in the matrix fraction was set to 1 and relative vector amounts of the depleted variants are plotted in figure 5 . upon depletion of the s /
mar with xmii the amount of pepi - egfp vector dna in the matrix fraction is decreased by 16% ( 0.84    0.1-fold over linearized pepi - egfp figure 5a ) . in contrast , the amount of pepi - hs4opp remained nearly unchanged upon depletion of the s / mar ( 0.96    0.13-fold over linearized pepi - hs4opp ) . to test whether the chs4 element is involved in the association with the nuclear matrix
, we released the chs4 element using xmai ; pepi - hs4opp vector dna was again traced in the matrix fraction . upon depletion of the s /
mar element the amount of pepi - hs4opp vector dna in the matrix fraction remained nearly unchanged ( 0.83-fold over linearized pepi - hs4opp ) , while there was a marked reduction of 35% of pepi - hs4opp dna after depletion of the chs4 element ( 0.65-fold over linearized pepi - hs4opp figure 5b ) .
using a similar approach , endogenous dna in nuclear matrix preparations was measured by preserving native attachment sites using cho / pepi - hs4opp#1 cells .
proteins were stripped from nuclei with a mild detergent and subsequently dna was digested with dnase i. remaining dna fragments were precipitated and used for quantitative pcr analyses with total dna extracted from cho / pepi - hs4opp#1 serving as reference and set arbitrarily to 1 . here ,
the s / mar sequence in pepi - hs4opp could be amplified , while the cmv promoter sequence was depleted by the digestion ( data not shown ; 3cmv 0.071    0.05 , 3s / mar 0.302    0.19 , p = 0.055 ) . within the 1.2  kb chs4 element , the 3hs4 fragment could be amplified , while the 5hs4 fragment was amplified at very low levels and apparently reduced by digestion ( data not shown ; 3hs4 0.29    0.35 , 5hs4 0.021    0.01 p = 0.18 ) . a protein that mediates many insulator functions and
is involved in a broad range of chromatin regulatory events is the ccctc - binding factor ( ctcf ) .
using chromatin immunoprecipitation ( chip ) , we examined binding of ctcf to pepi - hs4opp plasmid dna .
we detected a low signal for the 3cmv and 3s / mar region at background levels ( 1.85    2.5% of input dna ) , and a slightly increased signal for the 3hs4 region ( 2.5    0.44% of input dna ) .
for the 5hs4 region a distinct signal for ctcf binding was detectable ( 11.5    9% of input dna ; figure 6 ) .
the core unit being essential for its episomal behavior consists of an active transcription unit running into a scaffold / matrix attachment region ( s / mar ) . its nonviral origin and episomal behavior , even in the absence of selective pressure , predestine pepi as a promising alternative for gene therapeutic approaches .
however , a major factor limiting its use in gene therapy is the low efficiency with which the vector establishes as a replicating episomal entity following delivery into the desired target cells .
nearly 95% of all plasmids are lost during subsequent cell division with only 15% of the originally transfected vector molecules able to establish in the nucleus and stably replicate episomally . since the functionality of the vector depends on the nuclear compartment it reaches after transfection , it would be desirable to identify genomic sequences that are involved in genome organization and thus directing dna ( plasmid ) sequences into certain , preferentially active , nuclear compartments . within the last decade ,
multiple modifications of the prototypical pepi vector have been applied for its use in vivo and in vitro ( reviewed in ref . ) .
nevertheless , all these modifications provided little or no improvement in transgene expression , copy number per cell , and/or episomal establishment efficiency . in this study , we focused on improving pepi vector function with respect to transgene expression and episome establishment efficiency . by inserting cis - active sequences , namely the chs4 insulator and a ucoe , we demonstrated increased establishment via the chs4 element and increased transgene expression and copy number by utilizing the a2ucoe .
the a2ucoe region used in this study was derived from a methylation - free cpg island spanning the divergently transcribed promoters of the housekeeping genes hnrpa2b1 and cbx3 .
the a2ucoe is not a classical enhancer and was shown to confer resistance to silencing of the cmv promoter in a lentiviral vector system when inserted upstream the expression cassette . within recent years , the hnrpa2b1-cbx3a2ucoe region has been successfully employed in the construction of expression vectors that have markedly enhanced and improved the generation of cell lines for therapeutic protein production and retroviral / lentiviral vectors for human gene therapy applications .
consistent with these observations we detected increased egfp expression levels ( mean fi ) up to twofold over non - a2ucoe containing vectors ( pepi - egfp and pepi - hs4opp ; figure 2b , c ) , demonstrating an a2ucoe - mediated effect on gene expression in an episomal vector system .
our demonstration of a2ucoe functionality within a replicating episomal vector context suggests that this genomic element can not only confer resistance to silencing and elevated expression on silencing - prone viral promoters such as cmv used in this study , but also on other ubiquitously active or tissue specific promoters as has been demonstrated within lentiviral vectors .
however , we assumed that this increased expression is associated with histone modifications typical for actively transcribed chromatin .
we therefore performed chip analyses using antibodies against histone h3 and its counterpart acetylated at lysine residues 9 and 14 ( h3k9/14ac ) .
acetylated h3 is most often found in promoter / enhancer regions and h3k9/14ac was shown to be crucial for recruiting tfiid to ifn- locus .
we found both h3 and h3k9/14ac levels decreased in the cmv promoter and egfp coding region of pepi - ucoe when compared with pepi - egfp ( figure 4 ) , which was initially surprising .
however , since constantly active transcription is marked by an open chromatin structure , this might explain the overall reduced amount of histone h3 and thus a reduced level of h3k9/14ac .
consistently with this suggestion we found an increased level of h3k9/14ac in the 3a2ucoe region .
similar results were previously reported by lindahl allen and antoniou , showing that the endogenous hnrpa2b1-cbx3 a2ucoe region is characterized by an extended length of unmethylated dna contributing to its chromatin - opening function , which then promotes efficient transcription in association with elevated levels of active histone modification marks ( h3 acetylation ; h3k4 tri - methylation ) and overall depletion of nucleosomes at the cbx3 and hnrpa2b1 promoters .
another cis - acting element we functionally evaluated in pepi was the chs4 1.2  kb insulator element from the chicken -globin locus control region .
of increased homogeneity of transgene expression employing this element and since the chs4 is known to interact with the nuclear matrix , the chs4 insulator was inserted downstream and in opposite orientation with respect to the s / mar , resulting in pepi - hs4opp ( figure 1c ) .
in contrast to pepi - ucoe , cells transfected with pepi - hs4opp showed moderate transgene expression similar to cells transfected with pepi - egfp ( figure 2b , c ) , but a significantly elevated rate of episomal plasmid establishment ( figure 2a ) .
rate of establishment or establishment efficiency describes the frequency with which s / mar containing vector molecules are stably and episomally maintained after transfection and inherited during subsequent cell divisions , even in the absence of selective pressure .
we observed low establishment efficiencies for pepi - egfp ( 9.5    7.25% ) and pepi - ucoe ( 11.2    9.04% ) , meaning that ~90% of all initially transfected molecules are lost .
it is widely accepted that an active transcription running into the s / mar is mandatory for episomal maintenance of s / mar based vectors . but low establishment efficiencies of pepi - ucoe suggest that high transcription rates do not affect efficiency of the initial establishment event . however , pepi - hs4opp was found to establish with an increased efficiency of 25.3    13.8% ( p = 0.028 ) .
it has been shown that pepi plasmid vectors interact with the nuclear matrix via an s / mar  saf - a interaction and thereby are stably maintained .
furthermore , it is known that the chs4 insulator element associates with the nuclear matrix through its interaction with ctcf .
here we were able to demonstrate that : ( i ) pepi - egfp accumulated in the matrix fraction with depletion of the s / mar led to a reduction of pepi - egfp in the nuclear matrix fraction ( figure 5 ) ; ( ii ) pepi - hs4opp also accumulated in the matrix fraction but independently of the s / mar , while release of the 1.2  kb chs4 element led to a decrease of pepi - hs4opp in the matrix fraction ( figure 5 ) , and ( iii ) ctcf was found to associate with the 5 half of chs4 and with a somewhat weaker binding detectable in the 3 region ( figure 6 ) .
therefore , our data suggest that the elevated establishment efficiency of pepi - hs4opp is due to the presence of chs4 interacting with the matrix and thus increasing the probability of becoming  anchored  onto this nuclear structure and being established with higher efficiency .
however , since it has been convincingly described before that an ongoing transcription running into the s / mar is sufficient for episomal replication , we assume that a construct harboring the chs4 element alone would not be able to replicate episomally and thus would not be mitotically stable the absence of selection .
a pepi derivative combining both the a2ucoe and chs4 elements was also constructed and found to be maintained episomally , showing elevated ( a2ucoe - mediated ) egfp expression but did not establish with an efficiency comparable with that of pepi - hs4opp , containing chs4 alone ( data not shown ) , which may be due to its increased size ( ~11.5  kb ) . nevertheless ,
to date s / mar based vectors represent one , if not the only , attractive alternative to viral vectors for gene therapy applications . here
we show that the limiting factors of s / mar based vectors , low and/or unstable transgene expression and low replicating episomal establishment efficiency were markedly improved by insertion of chromosomal cis - acting elements .
the detailed functional mechanisms , especially those underlying the pepi - hs4opp  nuclear matrix interaction , need to be further investigated .
we believe that knowledge of the correlation between nuclear architecture , transgene expression , and plasmid establishment will have crucial impact on our understanding of nuclear organization and may also lead to improved vectors for nonviral gene therapy .
the functional element essential for episomal replication and maintenance of pepi is a promoter - transgene - s / mar unit .
the original pepi - egfp harbors a cmv - egfp - s / mar transcription unit . starting with the pepi(ucoe - hcr - intron - pdx ) , a kind gift from j. fabre ( king 's college london , london , uk ) , pepi - ucoe was constructed as follows : pepi(ucoe - hcr - intron - pdx ) , which contains the 1.5  kb core region of the hnrpa2b1-cbx3 ucoe was digested with asei to replace the hcr - intron - pdx unit by a cmv - egfp fragment from pepi - egfp ( figure 1a ) resulting in pepi - ucoe ( figure 1b ) .
pepi - hs4opp was constructed by cloning the 1.2  kb chs4 insulator fragment from pni - cd ( kindly provided by g. felsenfeld , nih , usa ) into pepi - egfp downstream the s / mar in opposite direction using the in - fusion cloning system ( clontech , st germain - en - laye , france ) and primers ss 5-tagatccggtggatccgagctcacggggacagcc-3 and as 5-cgcgggcccgggatccaatattctcactgactccgtcct-3. vectors , transfection and cell culture conditions .
the vectors pepi - egfp , pepi - hs4opp , and pepi - ucoe were transfected into cho cells ( german resource centre for biological material , dsmz , braunschweig , germany ) using fugene hd transfection reagent ( roche , mannheim , germany ) or amaxa cell line nucleofector kit t ( lonza , cologne , germany ) .
transfections were carried out with freshly prepared plasmid dna and only preparations with 90% supercoiled pdna were used for transfection .
cells were maintained in ham 's f12 medium ( pan biotech , passau , germany ) supplemented with 10% fetal bovine serum ( paa , pasching , germany ) , penicillin ( 10,000 units / ml)/streptomycin ( 10  mg / ml ) ( paa ) , and 50 g / ml partricin ( biochrom , berlin , germany ) . at 24 hours , post - transfection , transient transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis .
stably transfected cells were selected in the presence of 400 g / ml g418 for at least 14 days and plasmid maintenance of the pepi derivates was determined by flow cytometry analysis analyses over a time period of 60 days in the absence of selection .
transfected cells were rinsed with pbs to remove medium and centrifuged for 6 minutes at 666g .
analyses were performed with becton dickinson facscalibur multicolor flow cytometer ( bd biosciences , heidelberg , germany ) equipped with a 488  nm laser capable of detecting and distinguishing fluorescence emissions and bd cellquest analysis software .
the percentage of egfp positive cells as well as egfp fluorescence intensity of each sample was determined .
the bd cellquest analysis software provides values for the mean fi of egfp based on the number of fluorochromes contained in each cell captured by the flow cytometer .
therefore , the fi can be used as an indicator of transgene expression level in a distinct cell population .
colony - forming assay . to determine replicating episome establishment efficiency of the different pepi derivates ,
briefly , 24 hours post - transfection efficiency was determined as described above and 50 , 100 , and 200 egfp positive cells were seeded into a 150  mm cell culture dish in the presence of 400 g / ml g418 , respectively .
after 14 days of selection , g418 resistant colonies were fixed with 1% formaldehyde / pbs for 15 minutes at room temperature and subsequently stained with 0.5% crystal violet/25% methanol for 10 minutes at room temperature .
afterwards , plates were rinsed with ddh2o to remove excessive staining solution and colonies were counted . establishment efficiency of a respective vector was calculated using the ratio of resistant colonies to seeded cells .
total genomic dna or low molecular weight dna from a hirt extract was either isolated from a selected g418 resistant cell population or individual clones . in order to confirm replicative episomal maintenance of pepi constructs , total genomic dna or dna from a hirt extract
were codigested with either dpni and a plasmid single - cutter or mboi and a plasmid single - cutter ( pcii for pepi - egfp and pepi - ucoe ; hindiii for pepi - hs4opp ) .
digested dna was subjected to southern blot analysis as previously described using -p[dctp]-labeled linearized pepi plasmid dna lacking the s / mar element as a probe .
quantitative pcr analysis was performed using a light cycler instrument ( roche ) and the faststart dna master syber green i reaction mix ( roche ) .
pcr reactions were performed at 40 cycles using standard settings as recommended by the manufacturer .
plasmid copies were quantified using a single copy housekeeping gene ( dhfr ; ss 5-gttggaggcagttccgttta-3 as 5-agctgggataatgtgctgct-3 101  bp product ) as reference gene .
an aliquot of 10 cells per reaction were washed once with cold pbs followed by incubation in cytoskeleton buffer ( 10 mmol / l pipes , 300 mmol / l saccharose , 100 mmol / l nacl , 3 mmol / l mgcl2 , 1  mol / l egta ; 4 minutes on ice ) and centrifugation ( 1000g , 4 c , 3 minutes ) ; supernatant contained soluble cyto- and nucleoplasmic proteins .
cells were then incubated in extraction buffer ( 10 mmol / l pipes , 300 mmol / l saccharose , 250 mmol / l ammonium sulphate , 3 mmol / l mgcl2 , 1  mol / l egta ; 4 minutes on ice ) and collected by centrifugation as above ; supernatant contained soluble nuclear components including histone h1 .
digestion was performed in digestion buffer ( 10 mmol / l pipes , 300 mmol / l saccharose , 50 mmol / l nacl , 3 mmol / l mgcl2 , 1  mol / l egta ; 3 hours , 37 c ) using five enzymes that do not cut within pepi - egfp and pepi - hs4opp ( spei , noti , ecorv , xhoi , and pvui ) together with either ecori ( linearizing ) , xmii ( s / mar depletion ) , or xmai ( chs4 depletion ) .
subsequent centrifugation left the complete matrix in the pellet fraction while supernatant contained dna and histones .
after extraction with 2  mol / l nacl buffer ( 10 mmol / l pipes , 300 mmol / l saccharose , 2  mol / l nacl , 3 mmol / l mgcl2 , 1  mol / l egta ; 4 minutes on ice ) matrix - associated proteins , which are not part of the core filament were located in the supernatant ; matrix proteins , and the core filament network ( the  matrix fraction  ) were resuspended in 1 te/0.1% sds and subjected to proteinase k digestion , followed by phenol - chloroform extraction and dna precipitation .
primers used for quantitative pcr were derived from the neomycin gene ( ss 5-ggagaggctattcggctatgac-3 as 5-cgtcaagaaggcgatagaaggc-3 ) .
low ionic strength matrices from 10 cho cells ( cho / pepi - hs4opp#1 ) were prepared as described elsewhere .
briefly , cells were detached by trypsin treatment , washed twice in cold 1 pbs and resuspended in 8  ml isolation buffer ( 3.75 mmol / l tris - hcl , ph 7.4 ; 20 mmol / l kcl ; 0.5 mmol / l edta / koh ; 0.05 mmol / l spermidine , 0.125 mmol / l spermin ; 0.5% triton - x100 ; protease inhibitor cocktail ( roche ) ) .
subsequently , cells were homogenized by 15 strokes of a loose fitting pestle potter homogenizer .
nuclei were pelleted ( 900g , 10 minutes , 4 c ) and washed three - times in isolation buffer ; washed nuclei were resuspended in 600 l isolation buffer without edta and incubated for 20 minutes in a 37 c waterbath .
extraction buffer ( 5 mmol / l hepes / naoh , ph 7.4 ; 2 mmol / l kcl ; 2 mmol / l edta ; 0.25 mmol / l spermidine ; 0.5% triton - x100 ; 25 mmol / l lithium 3,5-diiodosalicylate ) was added slowly to a final volume of 7  ml .
after 5 minutes of incubation at room temperature , nuclei were recovered by centrifugation at 2400g , 20 minutes at room temperature and washed three - times in 1 digestion buffer ( 10 : 400
mmol / l tris - hcl , 100 mmol / l nacl , 60 mmol / l mgcl2 ; 10 mmol / l cacl2 ; ph 7.9 ) .
digestion with rnase - free dnase ( roche ) was performed with 100  u dnase for at least 3 hours at room temperature .
matrices were then centrifuged for 5 minutes at 1500g , washed four - times with digestion buffer , resuspended in 1 te/0.1% sds , and subjected to proteinase k digestion , followed by phenol - chloroform extraction and dna precipitation .
quantitative pcrs were performed using a light cycler instrument ( roche ) and the faststart dna master syber green i reaction mix ( roche ) .
following primer pairs were used : 3 cmv ( ss 5-tacatcaatgggcgtggata-3 , as 5-ggcggagttgttacgacatt-3 ) , 3 s / mar ( ss 5-acggcaaagccattttcata-3 , as 5-aaaaacatggaggagcgttg-3 ) , 3 hs4 ( ss 5-agcagtgtctgtggcctttt-3 , as 5-acctgttcagtgcgtcacc-3 ) , 5 hs4 ( ss 5-ttggatctggatgagcactg-3 , as 5-gggaggtgtgggaggttt-3 ) .
cross - linking of confluent cell cultures was performed with 1% paraformaldehyde / pbs ( ph 7.4 ) for 10 minutes at room temperature and stopped by adding glycine to a final concentration of 0.25  mol / l .
cells were washed twice with ice - cold pbs , scraped into a 15  ml tube and centrifuged ( 900g , 15 minutes , 4 c ) .
cells were then washed twice in triton - x buffer ( 0.25% triton - x100 ; 10 mmol / l edta ; 10 mmol / l tris / hcl , ph 8.1 ; 10 mmol / l nacl ; 1 protease inhibitor cocktail ( pic , roche ) ) , followed by resuspension in sds lysis buffer ( 1% sds ; 5 mmol / l edta ; 50 mmol
/ l tris / hcl , ph 8.1 ; 1 pic ( roche ) ) , and chromatin sheared to an average size of 2001200  bp by sonication on ice .
sonicated chromatin was centrifuged ( 14,000g , 15 minutes , 4 c ) and the supernatant used for immunoprecipitation .
protein g dyna beads ( invitrogen ) were prepared by washing twice with 0.1% triton - x100/pbs followed by two washing steps with tse i buffer ( 0.1% sds ; 1% triton - x100 ; 2 mmol / l edta ; 20
mmol / l tris / hcl , ph 8.1 ; 150 mmol / l nacl ; 1 pic ( roche ) ) . beads and respective antibodies ( 3 g )
were preincubated by rotation in 1  ml dilution buffer ( 1% triton - x100 ; 2 mmol / l edta ; 20 mmol / l
tris / hcl , ph 8.1 ; 150 mmol / l nacl ; 1 pic ( roche ) ) for 30 minutes at room temperature , followed by 2.5 hours at 4 c .
chromatin was then added and incubated over night at 4 c while mixing by rotation .
a specified aliquot of the nonprecipitated chromatin was saved to determine the quantity of input dna . on the following day beads
were washed once with dilution buffer , twice with ice - cold tse i buffer , twice with buffer iii ( 0.25 mmol / l licl ; 1% np-40 ; 1% deoxycholate ; 1 mmol / l edta ; 10 mmol / l tris / hcl , ph 8.1 ; 1 pic ( roche ) ) and once with te wash buffer ( 2 mmol / l edta ; 10
mmol / l tris / hcl , ph 8.1 ; 1 pic ( roche ) ) . between each washing step , beads were incubated rotation at 4 c for 10 minutes .
/ l edta ; 50 mmol / l tris / hcl , ph 8.1 ) and incubated for 5 hours at 65 c and 1400  rpm to reverse the paraformaldehyde crosslinks .
proteinase k digestion was then performed overnight at 55 c and immunoprecipitated dna extracted using phenol : chloroform extraction and ethanol precipitation .
subsequently , dna was disolvedin 50 l ddh2o , treated with rnase a , and subjected to quantitative pcr ( light cycler , roche ) .
the following antibodies were used for chip : anti - ctcf ( millipore , cat - no 17 - 10044 ) , anti - h3 ( abcam , ab1791 ) , and anti - h3k9/k14ac ( santa cruz , sc8655 ) .
following primers were used for quantitative pcr : 3 cmv ( ss 5-tacatcaatgggcgtggata-3 , as 5-ggcggagttgttacgacatt-3 ) , 3 s / mar ( ss 5-acggcaaagccattttcata-3 , as 5-aaaaacatggaggagcgttg-3 ) , 3 hs4 ( ss 5-agcagtgtctgtggcctttt-3 , as 5-acctgttcagtgcgtcacc-3 ) , 5 hs4 ( ss 5-ttggatctggatgagcactg-3 , as 5-gggaggtgtgggaggttt-3 ) , egfp - tss ( ss 5-tacatcaatgggcgtggata-3 , as 5-gtttacgtcgccgtccag-3 ) , 5 egfp ( ss 5-acgtaaacggccacaagttc-3 , as 5-aagtcgtgctgcttcatgt-3 ) , 3 egfp ( ss 5-cgaccactaccagcagaaca-3 , as 5-gaactccagcaggaccatgt-3 ) .
 the vector pepi was the first nonviral and episomally replicating vector . 
 its functional element is an expression unit linked to a chromosomal scaffold / matrix attached region ( s / mar ) . 
 the vector replicates autonomously with low copy number in various cell lines , is mitotically stable in the absence of selection over hundreds of generations , and was successfully used for the efficient generation of genetically modified pigs . since it is assumed that establishment of the vector is a stochastic event and 
 strongly depends on the nuclear compartment it reaches after transfection , it is of great interest to identify genomic sequences that guide dna sequences into certain nuclear compartments . 
 here we inserted genomic cis - acting sequences into pepi and examined their impact on transgene expression , long - term stability , and vector establishment . 
 we demonstrated that a ubiquitous chromatin - opening element ( ucoe ) mediated enhanced transgene expression , while an insulator sequence ( chs4 ) increased establishment efficiency , presumably via an additional interaction with the nuclear matrix . 
 thus , besides being a promising alternative to currently used viral vectors in gene therapeutic approaches , pepi may also serve as a tool to study nuclear compartmentalization ; identification of genomic cis - acting sequences that are involved in nuclear organization will contribute to our understanding of the interplay between transgene expression , plasmid establishment , and nuclear architecture . 
Introduction
Results
pEPI-UCOE and pEPI-HS4opp are maintained episomally and undergo cellular replication
A2UCOE and cHS4 increase transgene expression and establishment of the episomal vector pEPI
The cHS4 element interacts with the nuclear matrix enhancing S/MAR matrix interaction
Discussion
Materials and methods