BACKGROUND
over the last years the pig is getting more and more attractive as model organism for biomedical research due to similarities with humans in anatomy, size, physiology, metabolism and pathology .
early sexual maturity and large litter sizes make pigs more suitable for research than other larger animals like cow, sheep or dog .
furthermore, genetically engineered pigs are a promising source of cells, tissues and organs for xenotransplantation .
genetic modification of pigs can be achieved by a variety of approaches, including pronuclear microinjection of dna, sperm-mediated gene transfer, retroviral transduction, and somatic cell nuclear transfer  using transfected donor cells .
due to the lack of germ line competent pluripotent stem cells in pigs, the latter approach is currently the only route for the generation of gene targeted pigs  <cit> .
in addition, scnt using pools of stable transfected cell clones was an efficient way for the production of transgenic founder pigs with appropriate expression patterns  and facilitated the generation of the first pig models with inducible transgene expression  <cit> .
among the various parameters influencing the outcome of scnt, such as oocyte quality, nuclear transfer protocol, embryo culture or recipient animal preparation, the type and quality of the nuclear donor cells are of vital importance .
many primary cell lines established after passaging a primary culture were used for scnt in pigs, with differences regarding the cell type, source organ, age and gender of the donor animal.
among these cells, mesenchymal stem cells are more or less precisely characterized  <cit> .
fibroblast-like cells from fetuses, neonatal, juvenile or adult animals  <cit>  or unspecified cells from different organs  <cit>  are most widely used.
these cells have been tested for their efficiency in scnt experiments, but further characterization of the specific cell types, their morphology, proliferation, lifespan and stability of karyotype is mostly lacking.
the latter parameters are particularly important for advanced genetic modification technologies such as gene targeting, where transfected cells need to undergo homologous recombination, clonal selection and proliferation up to a scale that provides a sufficient number of cells for recombination analysis, cryopreservation and subsequent scnt experiments.
the efficiency of genetic modification of primary cells also depends on the effective introduction of dna vectors into the cells.
different methods for the transfection of primary mammalian cells have been described, including chemical  and physical methods  as well as viral transduction  <cit> , but their applicability depends on the cell type into which the dna has to be introduced.
here, we characterize for the first time primary kidney cells  isolated from kidney samples of juvenile pigs for their proliferation capacity, morphological appearance, stability of karyotype, and uptake of exogenous dna after application of different transfection methods.
furthermore, we show their potential to support development of scnt embryos in vitro and of viable genetically modified pigs after additive gene transfer and homologous recombination.
RESULTS
morphology and growth potential of pkcs compared to fibroblasts of different origin
initial morphologic characterization of cells was performed  <dig> h after isolation by bright field microscopy.
the cultures were usually 70–100% confluent and displayed a mixture of different cell morphologies.
to characterize pkc diversity, the two kidney cell lines pkcm and pkc <dig> were subcloned by generation of single cell clones at passage  <dig> and their morphology was evaluated  <dig> to  <dig> days later .
the cell clones differed in morphology , cell size, colony formation , colony compactness , growth rate and lifespan.
in parallel, we investigated porcine fetal fibroblasts  and ear fibroblasts  which also displayed morphological heterogeneity, though to a lesser extent than pkcs.
in the primary cell lines pkcm, pkc <dig>  pff <dig> and pef <dig>  the heterogeneous appearance diminished with increasing passage numbers and the culture became dominated by cells with spindle shaped fibroblast-like morphology .
in a pilot experiment, pkcs seeded on non-coated cell culture plates did not get properly confluent and rather grew in islands.
in contrast, on collagen-coated plates the cells grew evenly spread before reaching confluence .
for systematic investigation of the growth behavior of pkcs on different coatings, the proliferation of the primary cell lines pkcm and pkc <dig> was determined  <dig> h after seeding of different cell numbers  on gelatin-, collagen- or non-coated 96-well culture plates using an mtt based proliferation assay .
the collagen-coated plates notably promoted proliferation of the cells of both kidney cell lines, as well as pff and pef cells .
therefore, all further culture experiments were performed in collagen-coated dishes.
growth behavior and population doubling time of pkcm and pkc <dig> was determined and compared to pff <dig> and pef <dig> at passage 4– <dig>  growth curves were generated and population doubling time was calculated in the log phase .
after seeding, pkcm and in particular pkc <dig> cells started earlier to proliferate and showed a steeper growth curve than pff <dig> and pef <dig> cells.
in the exponential growth phase, between  <dig> and  <dig> h after seeding , the kidney cell lines exhibited the shortest population doubling time with  <dig>  h  and  <dig>  h , followed by pff <dig>  and pef <dig> .
in a long-term culture experiment, kidney cells were maintained for a high number of passages ‐ pkc <dig> cells at least up to passage  <dig> and pkcm up to passage  <dig> ‐ showing only slight signs of senescence, whereas pff and pef could hardly be passaged more than  <dig> times.
karyotype analysis  revealed 80% normal chromosome counts in pkcm at passage  <dig> and 74% in pkc <dig> at passage  <dig>  which was similar to the proportion of normal karyotypes in pff <dig>  and pef <dig>  determined at passage  <dig>  due to the fact that it is difficult to prepare appropriate amounts of metaphases from cells with higher passages and cells with more than  <dig> passages are not used for scnt in our lab we analyzed pff <dig> and pef <dig> at passage  <dig>
in vitro development of scnt embryos derived from pkcs, pffs, and pefs
in  <dig> independent scnt experiments pkcm, pff <dig> and pef <dig> were used as donor cells .
fusion rate and the proportion of development to blastocyst were significant higher with pkcm as donor cells compared to pef <dig>  the cell numbers of blastocysts derived from pkcm, pff <dig> and pef <dig> donor cells corresponded to  <dig> ± <dig> ,  <dig> ± <dig>  and  <dig> ± <dig> , respectively, and were not significantly different.
analysis of blastocysts on day  <dig>  values with different superscript were significantly different , sem = standard deviation.
degen.=degenerated.
genetic modification of primary pkcs
chemical transfection methods
lipofection of pkc <dig> resulted in transfection efficiencies of  <dig> to 52% using  <dig>  μg pmaxgfptm dna,  <dig>  to  <dig> μl plus reagent and a lipid to dna ratio of  <dig> : <dig> or 3: <dig> .
thereby, acceptable fluorescence intensities  were observed, although serious proportions of detached cells indicated a high number of dead cells  and the attached cells were strongly spindle-shaped/partially enlarged and showed vacuoles/lipids .
in the control approach  cells had a good quality  without vacuoles and no detached cells .
scale for the assessment of cell quality: 1- excellent to good; 2- no vacuoles and lipids detectable, cells are bit stretched; 3- partially vacuoles and lipids, stretched cells; 4- many vacuoles and lipids, stressed cells, partially enlarged cells; 5- a lot of large vacuoles and lipids, altered morphology and enlarged cells; scale for the assessment of cells in suspension: 0- none; 1- low number of cells; 2- a lot of cells; fluor.
= fluorescence.
the highest nanofection efficiencies  of pkc <dig> were obtained using  <dig> μg of dna and  <dig> ,  <dig>  or  <dig> μl of nanofectin .
in these approaches, cell quality  and the proportion of detached cells  were acceptable, but the fluorescence intensity was low when compared to lipofection .
the control approaches  resulted in excellent to good cell quality  and no cells in suspension .
scale for the assessment of cell quality: 1- excellent to good; 2- no vacuoles and lipids detectable, cells are bit stretched; 3- partially vacuoles and lipids, stretched cells; scale for the assessment of cells in suspension: 0- none; 1- low number of cells; 2- a lot of cells; fluor.
= fluorescence.
physical transfection methods
to obtain optimal electroporation conditions for pkc <dig>  different dna concentrations , resuspension buffers  and voltages  were tested .
the greatest transfection efficiencies  were determined when using  <dig> × <dig> cells  and 1× <dig> cells  at  <dig> v. fluorescence intensity was very good, but a high proportion of cells did not attach after electroporation and indicated a high rate of cell loss during this process .
attached cells showed signs of stress including extreme spindle-shaped morphology and vacuoles .
the control experiment  resulted in stressed cells exhibiting large spindle-shaped morphology , but a lower number of cells did not attach .
scale for the assessment of cell quality: 1- excellent to good; 2- cells are bit stretched/strongly fibroblast-like; 3- stretched cells with vacuoles; 4- very spindle-shaped morphology 5- extreme spindle-shaped cells with vacuoles; scale for the assessment of cells in suspension: 0- none; 1- low number of cells; 2- a lot of cells; fluor.
= fluorescence.
endotoxin-free purified dna was used; voltage: 230v.
optimal nucleofection parameters were ascertained in initial experiments by testing various programs offered by the supplier.
program u <dig> was the most efficient resulting in 63% transfection efficiency, good fluorescence intensity  and cells of good quality  using  <dig> × <dig> cells of pkc <dig>   <dig> μg dna and  <dig> different nucleofection programs .
therefore, u <dig> was used for further experiments using different cell numbers and quantity of dna.
best transfection efficiency , high fluorescence intensity , low number of cells in suspension  and good quality of cell morphology  was achieved using  <dig> × <dig> cells and  <dig> μg dna.
the control experiment  resulted in best cell quality  and a low number of cells in suspension .
in pkcm, another primary kidney cell line, transfection efficiencies between 49% and 66% and high fluorescence intensity  were determined using  <dig> × <dig> cells and  <dig> μg dna.
representative pictures of nucleofected pkcs after using  <dig> μg dna,  <dig> × <dig> cells and u <dig> are shown in figure  <dig>
scale for the assessment of cell quality: 1- excellent-good; 2- cells are bit stretched/strongly fibroblast-like; 3- stretched cells with vacuoles; 4- cells with very spindle-shaped morphology 5- extreme spindle-shaped with vacuoles; scale for the assessment of cells in suspension: 0- none; 1- low number of cells; 2- a lot of cells; fluor.
= fluorescence.
after testing of  <dig> different nucleofection programs, u <dig> was the most efficient program for pff <dig>  due to transfection efficiency of 53%, excellent cell quality  and low number of cells in suspension .
transfection of the primary cell line pef <dig> using  <dig> different nucleofection programs resulted in poor transfection efficiencies  and low fluorescence intensity .
among the different transfection methods used for transient transfection of pkc <dig>  nucleofection is the most suitable, since it is highly efficient and results in viable cells of good quality.
generation of transgenic animals using pkcs as donor cells
additive gene transfer and re-cloning
for the generation of transgenic animals by additive gene transfer , resulting in random integration of a gene construct into the genome, wild-type cells were transfected with a gene construct including the gene of interest and a resistance gene for the generation of stable transfected cell clones after antibiotic selection.
since a mixed population of transfected and selected cell clones was used for scnt, the generated animals had to be examined for transgenesis and expression level in the organs and tissues of interest.
the best expressing animals were used for breeding, for re-cloning or for a second transfection with further gene constructs to generate multi-transgenic animals.
in summary  <dig> different primary porcine kidney cell populations, including pkcm and pkc <dig>  isolated from neonatal to 13-month-old wild-type or transgenic pigs served as donor cells for scnt after one or two nucleofection rounds with  <dig> different gene constructs  <cit> .
in re-cloning experiments,  <dig> different pkc lines isolated from neonatal to 13-month-old cloned transgenic pigs without any further genetic modification served as donor cells for scnt  <cit> .
overall the litter rate and size were satisfying, where  <dig> embryo transfers after cloning of transfected cells resulted in  <dig> litters  with  <dig> animals and  <dig> embryo transfers after re-cloning gave rise to  <dig> litters  with  <dig> piglets.
hence, different primary kidney cells, including pkcm and pkc <dig>  were capable of producing genetically modified pigs by additive gene transfer.
the efficiency in transgenic pig delivery did not show significant differences between pkc <dig>  pkcm and the fibroblasts examined .
gene targeting
in contrast to additive gene transfer, where pools of stable transfected cell clones can be used for scnt, gene targeting requires clonal selection, which means the generation of individual cell clones and their analysis for homologous recombination.
figure 5b shows the sequential gene targeting of  <dig> alleles for the production of a homozygous knockout pig.
the pkcm line was used for targeting of  <dig> different loci  <cit> , using linearized bac-dna.
after a first round of transfection and selection, correctly targeted cell clones  were used for scnt.
in summary,  <dig> ets resulted in  <dig> litters  with a total of  <dig> animals  and  <dig> interrupted pregnancies  containing  <dig> fetuses.
pkcs from heterozygous animals were used for targeting of the second allele of the locus.
after transfer of embryos to  <dig> recipients  <dig> litters  with  <dig> offspring  were obtained.
thus, scnt using targeted pkcm clones resulted in acceptable pregnancy rates and litter sizes.
discussion
various types of primary transgenic or non-transgenic porcine cells were used as nuclear donors for the production of cloned embryos or piglets, but the characterization of the cells is normally restricted to the efficiency of transgenic animal production.
nonetheless, a comprehensive analysis of defined cell lines might facilitate choosing appropriate cell types for advanced transgenic strategies and improve the use of primary cells for the production of large animal models.
we used two different primary kidney cell lines and compared them with fetal or ear fibroblasts and observed that subclones of these cells differ in morphology and growth potential.
various groups verified different types of fibroblasts  and other cell types such as dendritic cells, macrophages and lymphocyte-like cells in kidney cell cultures .
in our hands, the diversity of cell morphologies decreased over several passages in all investigated cell lines, probably due to the culture conditions promoting proliferation of few fibroblast-like cell types and a different lifespan of cultured cell types.
regarding the growth potential, pkcs grew faster than fetal or ear fibroblasts, although the kidney cells were isolated from 3-month-old animals, whereas pff originated from a fetus and the pef from a neonatal ear.
the diversity of cell types in pkcs might influence the growth of cell population in a positive way due to factors which are produced by neighboring cells, e.g.
the extracellular matrix produced by renal fibroblasts  <cit> .
however, we showed that all investigated cells, including the kidney cells, grew better on coated compared to non-coated plates.
it has already been shown that collagen type i, which is part of the extracellular matrix, is a prevalent substrate for the culture of several cell types, including human skin fibroblasts  <cit>  and pffs  <cit> .
various methods have successfully been used to introduce exogenous dna into porcine fibroblasts.
during chemical transfection, we suggest that the vesicles were formed of big dna-lipid conglomerates and the vacuoles probably originate from cell stress during transfection.
overall, our lipofection results are very heterogeneous due to the numerous parameter combinations  which had to be tested.
the tested chemical transfection methods and in addition the conventional electroporation were not suitable for efficient transfection either due to low transfection efficiencies, formation of vacuoles and lipids or high toxicity.
nucleofection has been used for transfection of various cells  <cit> .
we achieved best results by nucleofection of pkcs with 70% to 89% transfection efficiency, which is comparable to the efficiency of 90% in pffs  <cit> .
in general, after nucleofection of all primary cell lines  we observed low cytotoxicity and only marginal changes of cell morphology.
this confirms partly previous studies using the nucleofection technology, showing no effects on cell properties, such as alteration of cell morphology, response to chemicals and pattern of gene expression  <cit> .
using pkcs as donor cells for scnt, the obtained blastocyst rate of 21% was higher in comparison to other reports showing a blastocyst rate of  <dig> to  <dig> % after 6– <dig> days  <cit>  of embryo in vitro culture.
the scnt with pffs as donor cells resulted in a blastocyst rate of  <dig> %.
in other studies the blastocyst rate using pffs ranged from  <dig>  to  <dig> %  <cit> , which is several times higher compared to our blastocyst rate.
nevertheless, it is difficult to compare cloning efficiencies between different laboratories, because a plethora of parameters influences the outcome of scnt experiments.
even cell cultures from the same fetus may differ in their development capability of embryos after scnt  <cit> .
moreover, the blastocyst rate is not compulsory meaningful for in vivo developmental competence, it rather gives a rough estimation of the principle applicability of a defined primary cell line as a donor for scnt.
in the last years, the generation of cloned transgenic pigs using nucleofected fibroblasts from fetal or ear tissue was quite successful in our lab  <cit> .
the usage pkcs for scnt after transfection and for re-cloning  <cit>  was in our hands satisfying and comparable to pff, whereas it has to be kept in mind that different gene constructs were used.
furthermore, we were able to target successfully  <dig> different loci in pkcs by homologous recombination  <cit>  and scnt with several of these correctly targeted cell clones resulted in litter rates of  <dig> to 50%.
rogers et al.
<cit>  verified that homologous recombination depends on donor cells, because they achieved targeting frequencies between  <dig>  to  <dig> % using various primary pff cultures prepared from different fetuses, but from same uterus at the same time  <cit> .
this underlines the importance of characterization of donor cell cultures provided for genetic modification and scnt.
beside the easy isolation procedure and good proliferation capacity of kidney cells it has to be mentioned that for the generation of multi-transgenic pigs and re-cloning kidney cells can be isolated after killing the animals for gene expression analysis anyway or from a biopsy taken from the living animal.
CONCLUSIONS
our porcine kidney cells are probably a mixture of different cell types showing better proliferation rate, growth capacity, transfection efficiency and blastocyst rate after scnt compared to pffs and pefs.
there is no evidence that this mixed population has a negative influence on transfection and further procedures which are necessary for transgenic pig production.
primary porcine kidney cells are highly suitable for additive gene transfer and gene targeting, and subsequent production of genetically modified pigs by scnt.
it has to be kept in mind that each primary cell culture originates from a different animal and preparation, and might show different properties.
however, our dataset includes kidney cells from different animals and preparations all showing the same basic characteristics.

 over the last years the pig is getting more and more attractive as model organism for biomedical research due to similarities with humans in anatomy, size, physiology, metabolism and pathology. early sexual maturity and large litter sizes make pigs more suitable for research than other larger animals like cow, sheep or dog. furthermore, genetically engineered pigs are a promising source of cells, tissues and organs for xenotransplantation. genetic modification of pigs can be achieved by a variety of approaches, including pronuclear microinjection of dna, sperm-mediated gene transfer, retroviral transduction, and somatic cell nuclear transfer using transfected donor cells. due to the lack of germ line competent pluripotent stem cells in pigs, the latter approach is currently the only route for the generation of gene targeted pigs. in addition, scnt using pools of stable transfected cell clones was an efficient way for the production of transgenic founder pigs with appropriate expression patterns and facilitated the generation of the first pig models with inducible transgene expression. among the various parameters influencing the outcome of scnt, such as oocyte quality, nuclear transfer protocol, embryo culture or recipient animal preparation, the type and quality of the nuclear donor cells are of vital importance. many primary cell lines established after passaging a primary culture were used for scnt in pigs, with differences regarding the cell type, source organ, age and gender of the donor animal. among these cells, mesenchymal stem cells are more or less precisely characterized. fibroblast-like cells from fetuses, neonatal, juvenile or adult animals or unspecified cells from different organs are most widely used. these cells have been tested for their efficiency in scnt experiments, but further characterization of the specific cell types, their morphology, proliferation, lifespan and stability of karyotype is mostly lacking. the latter parameters are particularly important for advanced genetic modification technologies such as gene targeting, where transfected cells need to undergo homologous recombination, clonal selection and proliferation up to a scale that provides a sufficient number of cells for recombination analysis, cryopreservation and subsequent scnt experiments. the efficiency of genetic modification of primary cells also depends on the effective introduction of dna vectors into the cells. different methods for the transfection of primary mammalian cells have been described, including chemical and physical methods as well as viral transduction, but their applicability depends on the cell type into which the dna has to be introduced. here, we characterize for the first time primary kidney cells isolated from kidney samples of juvenile pigs for their proliferation capacity, morphological appearance, stability of karyotype, and uptake of exogenous dna after application of different transfection methods. furthermore, we show their potential to support development of scnt embryos in vitro and of viable genetically modified pigs after additive gene transfer and homologous recombination.  morphology and growth potential of pkcs compared to fibroblasts of different origin initial morphologic characterization of cells was performed h after isolation by bright field microscopy. the cultures were usually 70–100% confluent and displayed a mixture of different cell morphologies. to characterize pkc diversity, the two kidney cell lines pkcm and pkc were subcloned by generation of single cell clones at passage and their morphology was evaluated to days later. the cell clones differed in morphology, cell size, colony formation, colony compactness, growth rate and lifespan. in parallel, we investigated porcine fetal fibroblasts and ear fibroblasts which also displayed morphological heterogeneity, though to a lesser extent than pkcs. in the primary cell lines pkcm, pkc pff and pef the heterogeneous appearance diminished with increasing passage numbers and the culture became dominated by cells with spindle shaped fibroblast-like morphology. in a pilot experiment, pkcs seeded on non-coated cell culture plates did not get properly confluent and rather grew in islands. in contrast, on collagen-coated plates the cells grew evenly spread before reaching confluence. for systematic investigation of the growth behavior of pkcs on different coatings, the proliferation of the primary cell lines pkcm and pkc was determined h after seeding of different cell numbers on gelatin-, collagen- or non-coated 96-well culture plates using an mtt based proliferation assay. the collagen-coated plates notably promoted proliferation of the cells of both kidney cell lines, as well as pff and pef cells. therefore, all further culture experiments were performed in collagen-coated dishes. growth behavior and population doubling time of pkcm and pkc was determined and compared to pff and pef at passage 4– growth curves were generated and population doubling time was calculated in the log phase. after seeding, pkcm and in particular pkc cells started earlier to proliferate and showed a steeper growth curve than pff and pef cells. in the exponential growth phase, between and h after seeding, the kidney cell lines exhibited the shortest population doubling time with h and h, followed by pff and pef. in a long-term culture experiment, kidney cells were maintained for a high number of passages ‐ pkc cells at least up to passage and pkcm up to passage ‐ showing only slight signs of senescence, whereas pff and pef could hardly be passaged more than times. karyotype analysis revealed 80% normal chromosome counts in pkcm at passage and 74% in pkc at passage which was similar to the proportion of normal karyotypes in pff and pef determined at passage due to the fact that it is difficult to prepare appropriate amounts of metaphases from cells with higher passages and cells with more than passages are not used for scnt in our lab we analyzed pff and pef at passage in vitro development of scnt embryos derived from pkcs, pffs, and pefs in independent scnt experiments pkcm, pff and pef were used as donor cells. fusion rate and the proportion of development to blastocyst were significant higher with pkcm as donor cells compared to pef the cell numbers of blastocysts derived from pkcm, pff and pef donor cells corresponded to ±, ± and ±, respectively, and were not significantly different. analysis of blastocysts on day values with different superscript were significantly different, sem = standard deviation. degen.=degenerated. genetic modification of primary pkcs chemical transfection methods lipofection of pkc resulted in transfection efficiencies of to 52% using μg pmaxgfptm dna, to μl plus reagent and a lipid to dna ratio of: or 3:. thereby, acceptable fluorescence intensities were observed, although serious proportions of detached cells indicated a high number of dead cells and the attached cells were strongly spindle-shaped/partially enlarged and showed vacuoles/lipids. in the control approach cells had a good quality without vacuoles and no detached cells. scale for the assessment of cell quality: 1- excellent to good; 2- no vacuoles and lipids detectable, cells are bit stretched; 3- partially vacuoles and lipids, stretched cells; 4- many vacuoles and lipids, stressed cells, partially enlarged cells; 5- a lot of large vacuoles and lipids, altered morphology and enlarged cells; scale for the assessment of cells in suspension: 0- none; 1- low number of cells; 2- a lot of cells; fluor. = fluorescence. the highest nanofection efficiencies of pkc were obtained using μg of dna and, or μl of nanofectin. in these approaches, cell quality and the proportion of detached cells were acceptable, but the fluorescence intensity was low when compared to lipofection. the control approaches resulted in excellent to good cell quality and no cells in suspension. scale for the assessment of cell quality: 1- excellent to good; 2- no vacuoles and lipids detectable, cells are bit stretched; 3- partially vacuoles and lipids, stretched cells; scale for the assessment of cells in suspension: 0- none; 1- low number of cells; 2- a lot of cells; fluor. = fluorescence. physical transfection methods to obtain optimal electroporation conditions for pkc different dna concentrations, resuspension buffers and voltages were tested. the greatest transfection efficiencies were determined when using × cells and 1× cells at v. fluorescence intensity was very good, but a high proportion of cells did not attach after electroporation and indicated a high rate of cell loss during this process. attached cells showed signs of stress including extreme spindle-shaped morphology and vacuoles. the control experiment resulted in stressed cells exhibiting large spindle-shaped morphology, but a lower number of cells did not attach. scale for the assessment of cell quality: 1- excellent to good; 2- cells are bit stretched/strongly fibroblast-like; 3- stretched cells with vacuoles; 4- very spindle-shaped morphology 5- extreme spindle-shaped cells with vacuoles; scale for the assessment of cells in suspension: 0- none; 1- low number of cells; 2- a lot of cells; fluor. = fluorescence. endotoxin-free purified dna was used; voltage: 230v. optimal nucleofection parameters were ascertained in initial experiments by testing various programs offered by the supplier. program u was the most efficient resulting in 63% transfection efficiency, good fluorescence intensity and cells of good quality using × cells of pkc μg dna and different nucleofection programs. therefore, u was used for further experiments using different cell numbers and quantity of dna. best transfection efficiency, high fluorescence intensity, low number of cells in suspension and good quality of cell morphology was achieved using × cells and μg dna. the control experiment resulted in best cell quality and a low number of cells in suspension. in pkcm, another primary kidney cell line, transfection efficiencies between 49% and 66% and high fluorescence intensity were determined using × cells and μg dna. representative pictures of nucleofected pkcs after using μg dna, × cells and u are shown in figure scale for the assessment of cell quality: 1- excellent-good; 2- cells are bit stretched/strongly fibroblast-like; 3- stretched cells with vacuoles; 4- cells with very spindle-shaped morphology 5- extreme spindle-shaped with vacuoles; scale for the assessment of cells in suspension: 0- none; 1- low number of cells; 2- a lot of cells; fluor. = fluorescence. after testing of different nucleofection programs, u was the most efficient program for pff due to transfection efficiency of 53%, excellent cell quality and low number of cells in suspension. transfection of the primary cell line pef using different nucleofection programs resulted in poor transfection efficiencies and low fluorescence intensity. among the different transfection methods used for transient transfection of pkc nucleofection is the most suitable, since it is highly efficient and results in viable cells of good quality. generation of transgenic animals using pkcs as donor cells additive gene transfer and re-cloning for the generation of transgenic animals by additive gene transfer, resulting in random integration of a gene construct into the genome, wild-type cells were transfected with a gene construct including the gene of interest and a resistance gene for the generation of stable transfected cell clones after antibiotic selection. since a mixed population of transfected and selected cell clones was used for scnt, the generated animals had to be examined for transgenesis and expression level in the organs and tissues of interest. the best expressing animals were used for breeding, for re-cloning or for a second transfection with further gene constructs to generate multi-transgenic animals. in summary different primary porcine kidney cell populations, including pkcm and pkc isolated from neonatal to 13-month-old wild-type or transgenic pigs served as donor cells for scnt after one or two nucleofection rounds with different gene constructs. in re-cloning experiments, different pkc lines isolated from neonatal to 13-month-old cloned transgenic pigs without any further genetic modification served as donor cells for scnt. overall the litter rate and size were satisfying, where embryo transfers after cloning of transfected cells resulted in litters with animals and embryo transfers after re-cloning gave rise to litters with piglets. hence, different primary kidney cells, including pkcm and pkc were capable of producing genetically modified pigs by additive gene transfer. the efficiency in transgenic pig delivery did not show significant differences between pkc pkcm and the fibroblasts examined. gene targeting in contrast to additive gene transfer, where pools of stable transfected cell clones can be used for scnt, gene targeting requires clonal selection, which means the generation of individual cell clones and their analysis for homologous recombination. figure 5b shows the sequential gene targeting of alleles for the production of a homozygous knockout pig. the pkcm line was used for targeting of different loci, using linearized bac-dna. after a first round of transfection and selection, correctly targeted cell clones were used for scnt. in summary, ets resulted in litters with a total of animals and interrupted pregnancies containing fetuses. pkcs from heterozygous animals were used for targeting of the second allele of the locus. after transfer of embryos to recipients litters with offspring were obtained. thus, scnt using targeted pkcm clones resulted in acceptable pregnancy rates and litter sizes. discussion various types of primary transgenic or non-transgenic porcine cells were used as nuclear donors for the production of cloned embryos or piglets, but the characterization of the cells is normally restricted to the efficiency of transgenic animal production. nonetheless, a comprehensive analysis of defined cell lines might facilitate choosing appropriate cell types for advanced transgenic strategies and improve the use of primary cells for the production of large animal models. we used two different primary kidney cell lines and compared them with fetal or ear fibroblasts and observed that subclones of these cells differ in morphology and growth potential. various groups verified different types of fibroblasts and other cell types such as dendritic cells, macrophages and lymphocyte-like cells in kidney cell cultures. in our hands, the diversity of cell morphologies decreased over several passages in all investigated cell lines, probably due to the culture conditions promoting proliferation of few fibroblast-like cell types and a different lifespan of cultured cell types. regarding the growth potential, pkcs grew faster than fetal or ear fibroblasts, although the kidney cells were isolated from 3-month-old animals, whereas pff originated from a fetus and the pef from a neonatal ear. the diversity of cell types in pkcs might influence the growth of cell population in a positive way due to factors which are produced by neighboring cells, e.g. the extracellular matrix produced by renal fibroblasts. however, we showed that all investigated cells, including the kidney cells, grew better on coated compared to non-coated plates. it has already been shown that collagen type i, which is part of the extracellular matrix, is a prevalent substrate for the culture of several cell types, including human skin fibroblasts and pffs. various methods have successfully been used to introduce exogenous dna into porcine fibroblasts. during chemical transfection, we suggest that the vesicles were formed of big dna-lipid conglomerates and the vacuoles probably originate from cell stress during transfection. overall, our lipofection results are very heterogeneous due to the numerous parameter combinations which had to be tested. the tested chemical transfection methods and in addition the conventional electroporation were not suitable for efficient transfection either due to low transfection efficiencies, formation of vacuoles and lipids or high toxicity. nucleofection has been used for transfection of various cells. we achieved best results by nucleofection of pkcs with 70% to 89% transfection efficiency, which is comparable to the efficiency of 90% in pffs. in general, after nucleofection of all primary cell lines we observed low cytotoxicity and only marginal changes of cell morphology. this confirms partly previous studies using the nucleofection technology, showing no effects on cell properties, such as alteration of cell morphology, response to chemicals and pattern of gene expression. using pkcs as donor cells for scnt, the obtained blastocyst rate of 21% was higher in comparison to other reports showing a blastocyst rate of to % after 6– days of embryo in vitro culture. the scnt with pffs as donor cells resulted in a blastocyst rate of %. in other studies the blastocyst rate using pffs ranged from to %, which is several times higher compared to our blastocyst rate. nevertheless, it is difficult to compare cloning efficiencies between different laboratories, because a plethora of parameters influences the outcome of scnt experiments. even cell cultures from the same fetus may differ in their development capability of embryos after scnt. moreover, the blastocyst rate is not compulsory meaningful for in vivo developmental competence, it rather gives a rough estimation of the principle applicability of a defined primary cell line as a donor for scnt. in the last years, the generation of cloned transgenic pigs using nucleofected fibroblasts from fetal or ear tissue was quite successful in our lab. the usage pkcs for scnt after transfection and for re-cloning was in our hands satisfying and comparable to pff, whereas it has to be kept in mind that different gene constructs were used. furthermore, we were able to target successfully different loci in pkcs by homologous recombination and scnt with several of these correctly targeted cell clones resulted in litter rates of to 50%. rogers et al. verified that homologous recombination depends on donor cells, because they achieved targeting frequencies between to % using various primary pff cultures prepared from different fetuses, but from same uterus at the same time. this underlines the importance of characterization of donor cell cultures provided for genetic modification and scnt. beside the easy isolation procedure and good proliferation capacity of kidney cells it has to be mentioned that for the generation of multi-transgenic pigs and re-cloning kidney cells can be isolated after killing the animals for gene expression analysis anyway or from a biopsy taken from the living animal.  our porcine kidney cells are probably a mixture of different cell types showing better proliferation rate, growth capacity, transfection efficiency and blastocyst rate after scnt compared to pffs and pefs. there is no evidence that this mixed population has a negative influence on transfection and further procedures which are necessary for transgenic pig production. primary porcine kidney cells are highly suitable for additive gene transfer and gene targeting, and subsequent production of genetically modified pigs by scnt. it has to be kept in mind that each primary cell culture originates from a different animal and preparation, and might show different properties. however, our dataset includes kidney cells from different animals and preparations all showing the same basic characteristics. 
compared with pffs and pefs, pkcs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after scnt and in vitro cultivation. among the four transfection methods tested with a gfp expression plasmid, best results were obtained with the nucleofectortm technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. in this study, we evaluate the potential of primary porcine kidney cells as cell source for scnt, including their proliferation capacity, transfection efficiency, and capacity to support full term development of scnt embryos after additive gene transfer or homologous recombination. however, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. several of those pigs originated from pkcs that underwent homologous recombination and antibiotic selection before scnt. the high proliferation capacity of pkcs facilitates the introduction of precise and complex genetic modifications in vitro. somatic cell nuclear transfer is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research.
 somatic cell nuclear transfer is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. however, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. in this study, we evaluate the potential of primary porcine kidney cells as cell source for scnt, including their proliferation capacity, transfection efficiency, and capacity to support full term development of scnt embryos after additive gene transfer or homologous recombination.  pkcs could be maintained in culture with stable karyotype for up to passages, whereas porcine fetal fibroblasts and porcine ear fibroblasts could be hardly passaged more than times. compared with pffs and pefs, pkcs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after scnt and in vitro cultivation. among the four transfection methods tested with a gfp expression plasmid, best results were obtained with the nucleofectortm technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. usage of genetically modified pkcs in scnt resulted in approximately piglets carrying at least one of different transgenes. several of those pigs originated from pkcs that underwent homologous recombination and antibiotic selection before scnt.  the high proliferation capacity of pkcs facilitates the introduction of precise and complex genetic modifications in vitro. pkcs are thus a valuable cell source for the generation of porcine biomedical models by scnt. pigprimary kidney cellsfibroblastsnuclear transfergenetic engineering 