BACKGROUND
mouse embryonic stem  cells are derived from the inner cell mass  of the mouse blastocyst and retain pluripotency when cultured in vitro in the presence of factors that inhibit differentiation, such as leukaemia inhibitory factor .
however, when these factors are withdrawn, es cells have the potential to differentiate into ectodermal, mesodermal, endodermal and germ cell lineages .
es cells can differentiate either in suspension, forming embryoid bodies  or as two-dimensional adhesion cultures, either in the presence of feeder cells or extracellular matrix  molecules  <cit> .
the regulation of es cell differentiation is currently under intense study, not least for potential therapeutic use.
one important area of investigation is es cell differentiation either in ebs or in 2-dimensional culture, along pathways recapitulating vasculogenesis and early angiogenesis  <cit> .
related to this is delineation of functions for growth factors, adhesion molecules and transcription factors in the formation of the vasculature.
elucidating the role of angiogenic factors during differentiation often requires over-expression of wild type and mutant proteins.
transfection of es cells with plasmids using traditional electroporation and lipofectamine-based methods can be problematic  <cit> .
conflicting data about the activity of different promoters in undifferentiated es cells have also been obtained, while several studies have suggested that transcriptional inactivation of promoters occurs during selection of stably transfected cells  <cit> .
another problem is obtaining stable transgene expression through stem cell differentiation.
several studies have looked at the efficacy of promoters in plasmid based vector systems with a view to obtaining high transient and stable expression in stem cells.
for example, the cag promoter has been used to express transgenes in undifferentiated es cells as well as es cells undergoing differentiation towards neuronal, myogenic and mesodermal cell types  <cit> .
in the latter case a transfection system was used  leading to episomal propagation of the vector carrying the transgene of interest  <cit> .
although episomal plasmid propagation may lead to very high levels of transgene expression due to a high copy number of plasmid per cell, certain experiments may require lower levels of overexpression that may be more physiologically relevant.
here, we compare the promoter activity of the cmv, chicken β-actin and cag promoters in undifferentiated murine embryonic cce cells as well as during differentiation to mesoderm.
these cells are routinely used for differentiation into mesodermal lineages  <cit>  and do not express the polyoma virus large t antigen allowing integration of transgenes into the host's genome.
we demonstrate that of these vector systems, only plasmids containing the cag promoter can be used for long term expression of genes during differentiation of two dimensional cultures of mouse embryonic cce es cells grown on collagen substrates towards mesoderm.
RESULTS
differentiation of mesodermal cell lineages from mouse cce es cells
cells of a mesodermal/endothelial lineage were differentiated from mouse cce embryonic stem cells following the protocol described in  <cit>  .
undifferentiated cce cells expressed high levels of e-cadherin, ssea- <dig> and pecam- <dig>  which is consistent with previous data  <cit> .
these cells did not express the mesodermal/endothelial marker flk <dig>  the endothelial cell  marker ve-cadherin or the smooth muscle cell /pericyte marker pdgfrβ .
differentiation was initiated by the removal of lif from the medium and the seeding of cells on collagen type iv.
flk <dig> expression was observed on days 4– <dig> of differentiation, while elevated levels of pdgfrβ were also noted at the same time.
expression of pecam- <dig> was much reduced during differentiation and the levels of e-cadherin and ssea- <dig> also declined by day  <dig>  ve-cadherin levels remained negligible during this  <dig> day differentiation period .
flk1+ cells from either day  <dig> or day  <dig> cultures were facs sorted and replated on collagen type iv-coated plates in the presence of vegfa for a further  <dig> days .
double immunofluorescence microscopy for the ec markers ve-cadherin or pecam- <dig> and the smc marker α-smooth muscle actin   showed the presence of ec sheets surrounded by smcs.
differentiation into ecs was vegfa-dependent, as only αsma+ cells were observed in its absence .
when flk1+ cells were facs sorted and plated in three dimensional collagen type i gels in the presence of vegfa for an additional  <dig> days, the formation of angiogenic sprouts was observed .
tubular structures positive for the ec marker pecam- <dig> were surrounded by αsma+ smcs/pericytes .
the formation of tubes was dependent on vegfa/flk <dig> signalling, since no tubes formed in the absence of vegfa or when flk1- cells were sorted and cultured in the presence of vegfa .
these data are in agreement with previous studies  <cit>  and suggest that this differentiation protocol mimics aspects of both vasculogenesis and early angiogenesis and is suitable for ectopic expression studies.
the cmv and β-actin promoters are not suitable for sustained expression of transgenes during cce cell differentiation
high levels of transfection efficiency  of cce cells  were obtained with a modified lipofectamine based protocol where suspended cells were transfected with pegfp-n <dig>  <cit> .
this compared favourably with other methods such as electroporation  and the standard lipofectamine protocol .
however the longevity of gfp expression in transfected cce cells was poor.
after  <dig> days in culture under antibiotic selection very few fluorescing cells were evident  as compared to cultures  <dig> days after transfection .
these results were confirmed by facs analysis showing that only 34–50% of undifferentiated cells surviving antibiotic selection expressed gfp , while a dramatic decrease in the transgene's expression was noted in stably transfected cells differentiated for  <dig> days on collagen type iv to the flk <dig> stage .
it should be noted that at this stage the pool of cells contain both flk1+ and flk1- cells.
the gfp cdna in pegfp-n <dig> is under the control of the cmv promoter and it is not uncommon for transcriptional inactivation of this promoter to occur in certain cell types.
to overcome this problem we expressed gfp under the control of the chicken β-actin promoter.
gfp expression in differentiated cells was again very low  and this promoter offered no advantage over the cmv promoter.
since a higher percentage of transiently transfected cells expressed gfp compared to the stably transfected cells , cells  <dig> days post transfection were used for differentiation to test whether this would lead to more gfp+ cells present at the flk <dig> stage.
however, the use of transiently transfected cells resulted in no cells expressing gfp on day  <dig> of differentiation .
this suggested that stable integration of the transgene into the host's chromosome is required for its expression.
from the above studies it was evident that resistance against antibiotic selection did not correlate with expression of the transgene of interest.
therefore, bicistronic vectors having gfp as the reporter gene were tested, as they would allow for easy identification and selection of cells expressing the transgene of interest.
cce cells were transfected with either the pires2-egfp plasmid or pires2-egfp, where the cmv promoter had been substituted for the chicken β-actin promoter.
however, gfp expression was not apparent in cce cells transfected with either plasmid even  <dig> day after transfection .
control cultures were transfected in parallel with pegfp-n <dig> and gfp+ cells were abundant in this case.
the bicistronic vectors were then transfected into fibroblasts to check their functionality and fluorescing cells were obtained from transfections with both pires2-egfp and pires2-egfp .
the above data suggest that to obtain sustained transgene expression in murine cce cells and during differentiation into mesoderm, alternative promoters to the cmv and the chicken β-actin promoters are required.
furthermore, the above promoters driving expression in a vector that contains an ires element were inactive in undifferentiated es cells, in agreement with previous reports  <cit> .
vectors containing the cag promoter can be used to express transgenes in murine cce cells and during differentiation into mesoderm
the cmv immediate enhancer/β-actin  promoter has been shown to give higher levels of transgene expression in several cell lines compared to the cmv and β-actin promoters  <cit> .
two different vectors containing the cag promoter were used.
the first, pcagipuro is a bicistronic vector, where a puromycin resistance gene follows the ires element.
gfp was cloned into this vector to create pcagipuro-gfp .
the pcaggs plasmid is a monocistronic vector with gfp expression also under the control of the cag promoter.
the g <dig> resistance cassette from vector pmc1neo polya was cloned into pcaggs to create pcaggsneo-gfp .
cce cells were transfected with pcagipuro-gfp and selected for puromycin resistance.
facs analysis for gfp expression showed that virtually 100% of either undifferentiated  or cells differentiated to the flk <dig> stage  expressed high levels of gfp.
indeed, all of the cells in these differentiated cultures consisting of both flk1+ and flk1- cells were expressing gfp and this suggests that the cag promoter is active in both flk1+ and flk1- cells.
cce cells were also transfected with the pcaggsneo-gfp vector and were selected for g <dig> resistance.
when stable transfectants were analysed by facs, approximately 50% of either undifferentiated  or differentiated  cells were gfp+.
while this was a substantially lower percentage of positive cells than that obtained with the pcagipuro-gfp vector, it was much superior to the pegfp-n <dig> and plk444-gfp vectors, particularly during differentiation .
having shown that the pcagipuro vector could be used to achieve sustained gfp expression during differentiation of cce cells to mesodermal cell types we tested this vector system for its ability to express genes of biological interest.
the syndecans are type i transmembrane proteoglycans involved in cell adhesion, migration and growth factor interactions  <cit> .
in mammals there are four family members and we analysed undifferentiated and differentiating cce cells for syndecan expression using rt-pcr.
undifferentiated cce cells expressed only syndecans- <dig> and - <dig> , while flk1+ and flk1- cells facs sorted on day  <dig> of differentiation expressed all four syndecans .
we then decided to study syndecans- <dig> and - <dig> in further detail as they have been implicated in playing a role in the vascular system  <cit> .
facs analysis of undifferentiated cce cells confirmed that cell surface syndecan- <dig> is present in these cells whereas syndecan- <dig> is not .
it was only after differentiation that syndecan- <dig> appeared on the cell surface at low levels.
we then used the pcagipuro plasmid to express syndecans- <dig> and - <dig>  transfected cells were selected for puromycin resistance and the cells were differentiated on collagen type iv for  <dig> days.
facs analysis confirmed that high levels of cell surface expression of both proteins was achieved in undifferentiated as well as differentiated cells , although overexpression of these receptors had no effect on the percentage of flk1+ cells obtained during differentiation .
CONCLUSIONS
here we optimised a differentiation system of es cells into flk1+ mesodermal cells, ecs and smcs and subsequently used the cells to form angiogenic sprouts in vitro.
we then evaluated a number of plasmid vector systems for expressing transgenes during differentiation to flk1+mesodermal cells and found that vectors which utilise the cag promoter are most effective at driving gene expression and do not compromise the ability of stem cells to differentiate.
the cag promoter provides long term expression of transgenes in these cells however the system is most effective when the cells can be put under a selective pressure to express the transgene.
for this reason bicistronic vectors containing an antibiotic resistance gene offers the best way of obtaining expression of transgenes during differentiation into mesodermal flk1+ cells.
this is in contrast to vectors containing the cmv and β-actin promoters which gave very low levels of expression.
this presents a very useful tool for use in studies of angiogenesis and vasculogenesis.

 mouse embryonic stem cells are derived from the inner cell mass of the mouse blastocyst and retain pluripotency when cultured in vitro in the presence of factors that inhibit differentiation, such as leukaemia inhibitory factor. however, when these factors are withdrawn, es cells have the potential to differentiate into ectodermal, mesodermal, endodermal and germ cell lineages. es cells can differentiate either in suspension, forming embryoid bodies or as two-dimensional adhesion cultures, either in the presence of feeder cells or extracellular matrix molecules. the regulation of es cell differentiation is currently under intense study, not least for potential therapeutic use. one important area of investigation is es cell differentiation either in ebs or in 2-dimensional culture, along pathways recapitulating vasculogenesis and early angiogenesis. related to this is delineation of functions for growth factors, adhesion molecules and transcription factors in the formation of the vasculature. elucidating the role of angiogenic factors during differentiation often requires over-expression of wild type and mutant proteins. transfection of es cells with plasmids using traditional electroporation and lipofectamine-based methods can be problematic. conflicting data about the activity of different promoters in undifferentiated es cells have also been obtained, while several studies have suggested that transcriptional inactivation of promoters occurs during selection of stably transfected cells. another problem is obtaining stable transgene expression through stem cell differentiation. several studies have looked at the efficacy of promoters in plasmid based vector systems with a view to obtaining high transient and stable expression in stem cells. for example, the cag promoter has been used to express transgenes in undifferentiated es cells as well as es cells undergoing differentiation towards neuronal, myogenic and mesodermal cell types. in the latter case a transfection system was used leading to episomal propagation of the vector carrying the transgene of interest. although episomal plasmid propagation may lead to very high levels of transgene expression due to a high copy number of plasmid per cell, certain experiments may require lower levels of overexpression that may be more physiologically relevant. here, we compare the promoter activity of the cmv, chicken β-actin and cag promoters in undifferentiated murine embryonic cce cells as well as during differentiation to mesoderm. these cells are routinely used for differentiation into mesodermal lineages and do not express the polyoma virus large t antigen allowing integration of transgenes into the host's genome. we demonstrate that of these vector systems, only plasmids containing the cag promoter can be used for long term expression of genes during differentiation of two dimensional cultures of mouse embryonic cce es cells grown on collagen substrates towards mesoderm.  differentiation of mesodermal cell lineages from mouse cce es cells cells of a mesodermal/endothelial lineage were differentiated from mouse cce embryonic stem cells following the protocol described in. undifferentiated cce cells expressed high levels of e-cadherin, ssea- and pecam- which is consistent with previous data. these cells did not express the mesodermal/endothelial marker flk the endothelial cell marker ve-cadherin or the smooth muscle cell /pericyte marker pdgfrβ. differentiation was initiated by the removal of lif from the medium and the seeding of cells on collagen type iv. flk expression was observed on days 4– of differentiation, while elevated levels of pdgfrβ were also noted at the same time. expression of pecam- was much reduced during differentiation and the levels of e-cadherin and ssea- also declined by day ve-cadherin levels remained negligible during this day differentiation period. flk1+ cells from either day or day cultures were facs sorted and replated on collagen type iv-coated plates in the presence of vegfa for a further days. double immunofluorescence microscopy for the ec markers ve-cadherin or pecam- and the smc marker α-smooth muscle actin showed the presence of ec sheets surrounded by smcs. differentiation into ecs was vegfa-dependent, as only αsma+ cells were observed in its absence. when flk1+ cells were facs sorted and plated in three dimensional collagen type i gels in the presence of vegfa for an additional days, the formation of angiogenic sprouts was observed. tubular structures positive for the ec marker pecam- were surrounded by αsma+ smcs/pericytes. the formation of tubes was dependent on vegfa/flk signalling, since no tubes formed in the absence of vegfa or when flk1- cells were sorted and cultured in the presence of vegfa. these data are in agreement with previous studies and suggest that this differentiation protocol mimics aspects of both vasculogenesis and early angiogenesis and is suitable for ectopic expression studies. the cmv and β-actin promoters are not suitable for sustained expression of transgenes during cce cell differentiation high levels of transfection efficiency of cce cells were obtained with a modified lipofectamine based protocol where suspended cells were transfected with pegfp-n. this compared favourably with other methods such as electroporation and the standard lipofectamine protocol. however the longevity of gfp expression in transfected cce cells was poor. after days in culture under antibiotic selection very few fluorescing cells were evident as compared to cultures days after transfection. these results were confirmed by facs analysis showing that only 34–50% of undifferentiated cells surviving antibiotic selection expressed gfp, while a dramatic decrease in the transgene's expression was noted in stably transfected cells differentiated for days on collagen type iv to the flk stage. it should be noted that at this stage the pool of cells contain both flk1+ and flk1- cells. the gfp cdna in pegfp-n is under the control of the cmv promoter and it is not uncommon for transcriptional inactivation of this promoter to occur in certain cell types. to overcome this problem we expressed gfp under the control of the chicken β-actin promoter. gfp expression in differentiated cells was again very low and this promoter offered no advantage over the cmv promoter. since a higher percentage of transiently transfected cells expressed gfp compared to the stably transfected cells, cells days post transfection were used for differentiation to test whether this would lead to more gfp+ cells present at the flk stage. however, the use of transiently transfected cells resulted in no cells expressing gfp on day of differentiation. this suggested that stable integration of the transgene into the host's chromosome is required for its expression. from the above studies it was evident that resistance against antibiotic selection did not correlate with expression of the transgene of interest. therefore, bicistronic vectors having gfp as the reporter gene were tested, as they would allow for easy identification and selection of cells expressing the transgene of interest. cce cells were transfected with either the pires2-egfp plasmid or pires2-egfp, where the cmv promoter had been substituted for the chicken β-actin promoter. however, gfp expression was not apparent in cce cells transfected with either plasmid even day after transfection. control cultures were transfected in parallel with pegfp-n and gfp+ cells were abundant in this case. the bicistronic vectors were then transfected into fibroblasts to check their functionality and fluorescing cells were obtained from transfections with both pires2-egfp and pires2-egfp. the above data suggest that to obtain sustained transgene expression in murine cce cells and during differentiation into mesoderm, alternative promoters to the cmv and the chicken β-actin promoters are required. furthermore, the above promoters driving expression in a vector that contains an ires element were inactive in undifferentiated es cells, in agreement with previous reports. vectors containing the cag promoter can be used to express transgenes in murine cce cells and during differentiation into mesoderm the cmv immediate enhancer/β-actin promoter has been shown to give higher levels of transgene expression in several cell lines compared to the cmv and β-actin promoters. two different vectors containing the cag promoter were used. the first, pcagipuro is a bicistronic vector, where a puromycin resistance gene follows the ires element. gfp was cloned into this vector to create pcagipuro-gfp. the pcaggs plasmid is a monocistronic vector with gfp expression also under the control of the cag promoter. the g resistance cassette from vector pmc1neo polya was cloned into pcaggs to create pcaggsneo-gfp. cce cells were transfected with pcagipuro-gfp and selected for puromycin resistance. facs analysis for gfp expression showed that virtually 100% of either undifferentiated or cells differentiated to the flk stage expressed high levels of gfp. indeed, all of the cells in these differentiated cultures consisting of both flk1+ and flk1- cells were expressing gfp and this suggests that the cag promoter is active in both flk1+ and flk1- cells. cce cells were also transfected with the pcaggsneo-gfp vector and were selected for g resistance. when stable transfectants were analysed by facs, approximately 50% of either undifferentiated or differentiated cells were gfp+. while this was a substantially lower percentage of positive cells than that obtained with the pcagipuro-gfp vector, it was much superior to the pegfp-n and plk444-gfp vectors, particularly during differentiation. having shown that the pcagipuro vector could be used to achieve sustained gfp expression during differentiation of cce cells to mesodermal cell types we tested this vector system for its ability to express genes of biological interest. the syndecans are type i transmembrane proteoglycans involved in cell adhesion, migration and growth factor interactions. in mammals there are four family members and we analysed undifferentiated and differentiating cce cells for syndecan expression using rt-pcr. undifferentiated cce cells expressed only syndecans- and -, while flk1+ and flk1- cells facs sorted on day of differentiation expressed all four syndecans. we then decided to study syndecans- and - in further detail as they have been implicated in playing a role in the vascular system. facs analysis of undifferentiated cce cells confirmed that cell surface syndecan- is present in these cells whereas syndecan- is not. it was only after differentiation that syndecan- appeared on the cell surface at low levels. we then used the pcagipuro plasmid to express syndecans- and - transfected cells were selected for puromycin resistance and the cells were differentiated on collagen type iv for days. facs analysis confirmed that high levels of cell surface expression of both proteins was achieved in undifferentiated as well as differentiated cells, although overexpression of these receptors had no effect on the percentage of flk1+ cells obtained during differentiation.  here we optimised a differentiation system of es cells into flk1+ mesodermal cells, ecs and smcs and subsequently used the cells to form angiogenic sprouts in vitro. we then evaluated a number of plasmid vector systems for expressing transgenes during differentiation to flk1+mesodermal cells and found that vectors which utilise the cag promoter are most effective at driving gene expression and do not compromise the ability of stem cells to differentiate. the cag promoter provides long term expression of transgenes in these cells however the system is most effective when the cells can be put under a selective pressure to express the transgene. for this reason bicistronic vectors containing an antibiotic resistance gene offers the best way of obtaining expression of transgenes during differentiation into mesodermal flk1+ cells. this is in contrast to vectors containing the cmv and β-actin promoters which gave very low levels of expression. this presents a very useful tool for use in studies of angiogenesis and vasculogenesis. 
mouse embryonic stem cells cultured in vitro have the ability to differentiate into cells of the three germ layers as well as germ cells. cce mouse embryonic stem cells were differentiated on collagen type iv for 4– days, flk1+ mesodermal cells were sorted and replated either on collagen type iv in the presence of vegfa to give rise to endothelial cells and smooth muscle cells or in collagen type i gels for the formation of vascular tubes. the activity of the cmv and β-actin promoters was downregulated during selection of stable transfectants and during differentiation to the flk stage, while the cmv immediate enhancer/β-actin promoter in the pcagipuro-gfp vector led to 100% of stably transfected undifferentiated and differentiated cells expressing gfp. embryonic stem cells are difficult to transfect, while downregulation of promoter activity upon selection of stable transfectants has been reported, rendering the study of proteins by overexpression difficult. vectors containing the cag promoter offer a valuable tool for the long term expression of transgenes during stem cell differentiation towards mesoderm, while the cmv and β-actin promoters lead to very poor transgene expression during this process. to further test this system we expressed syndecan- and - in these cells and demonstrated high levels of transgene expression in both undifferentiated cells and cells differentiated to the flk stage.
 mouse embryonic stem cells cultured in vitro have the ability to differentiate into cells of the three germ layers as well as germ cells. the differentiation mimics early developmental events, including vasculogenesis and early angiogenesis and several differentiation systems are being used to identify factors that are important during the formation of the vascular system. embryonic stem cells are difficult to transfect, while downregulation of promoter activity upon selection of stable transfectants has been reported, rendering the study of proteins by overexpression difficult.  cce mouse embryonic stem cells were differentiated on collagen type iv for 4– days, flk1+ mesodermal cells were sorted and replated either on collagen type iv in the presence of vegfa to give rise to endothelial cells and smooth muscle cells or in collagen type i gels for the formation of vascular tubes. the activity of the cmv and β-actin promoters was downregulated during selection of stable transfectants and during differentiation to the flk stage, while the cmv immediate enhancer/β-actin promoter in the pcagipuro-gfp vector led to 100% of stably transfected undifferentiated and differentiated cells expressing gfp. to further test this system we expressed syndecan- and - in these cells and demonstrated high levels of transgene expression in both undifferentiated cells and cells differentiated to the flk stage.  vectors containing the cag promoter offer a valuable tool for the long term expression of transgenes during stem cell differentiation towards mesoderm, while the cmv and β-actin promoters lead to very poor transgene expression during this process. 