BACKGROUND
primordial germ cells , the precursor of gametes, have a unique migration activity in birds as well as in mammals.
they temporally reside in the extra-embryonic tissue and localize into embryonic gonads.
pgcs in mammals are originally derived from the epiblast of the gastrulating embryo and move into embryonic gonads through hindgut by amoeboid movement  <cit> .
in contrast, in birds, pgcs firstly appear from the epiblast in the blastoderm and translocate to the hypoblast of the area pellucida  <cit> .
during the gastrulation, they circulate through the vascular system and shuttle down into the gonadal anlagen  <cit> .
thus, avian pgcs can be collected from germinal crescent  <cit>  or blood vessel  <cit> , and embryonic gonads  <cit> .
recently, this unique migration pattern of avian pgcs allowed producing germline chimeras by re-transplantation of the pgcs into the blood vessel of recipient embryos  <cit> .
morphological and physiological features of avian germ cells including pgcs have been well characterized and utilized for further studies.
however, there are only a few reports on expressed sequence tag  analysis and functional genomic study for avian germ cells, especially pgcs in the early embryonic developmental stages.
pgc is an important cell type, in which either gene expression or suppression should be regulated temporally and spatially during embryonic developments.
according to gene expression switching triggered by interactions with environmental niche, pgcs could maintain their pluripotency or differentiate into germ cells.
however, due to technical difficulties, no further progress has been made in functional genomic study and massive novel gene mining in avian pgcs as well as neighboring stroma cells.
therefore, this study was conducted to investigate a large-scale est analysis in chicken macs-separated pgcs and compared the expression of the pgc ests with that of embryonic gonad.
RESULTS
retrieval of chicken pgcs by macs treatment
embryonic gonads were retrieved from total  <dig>  white leghorn  embryos at  <dig>  days of incubation and then the retrieved gonadal cells were treated with magnetic activated cell sorter  for separation of pgcs in total embryonic gonadal cells.
pgc population ratio after macs separation increased  <dig>  folds than that before macs .
thus, we collected  <dig>  ×  <dig> pgcs from  <dig>  embryos for total rna preparation and cdna library construction.
the population ratio of chicken pgcs in embryonic gonads was approximately  <dig> %  <cit> .
so, it is difficulty and complicated to retrieve a large number of chicken pgcs at the early embryonic stages.
thus, in this study, gene expression profiling of chicken pgcs was conducted and analyzed with macs-separated chicken pgcs.
the morphological and physiological properties of pgcs were unchanged even after macs separation  <cit> .
macs-separated pgcs has the reactivity to germ cell-specific antibodies and the migration capacity into embryonic gonads after re-transplantation into the recipient embryos  <cit> .
cdna library construction from chicken pgcs and est sequencing
after in vivo excision with e. coli strain solr, insert sizes of the cdna libraries from pgcs were analyzed by pcr and insert fragments ranged from  <dig>  to  <dig> kb .
titer of primarily cdna library was approximately  <dig>  ×  <dig> pfu/ml on average.
subsequently, we massively sequenced ests from pgc cdna library and total  <dig>  ests were sequenced.
of  <dig>  ests,  <dig> sequences were excluded due to low sequencing quality and after vector sequence trimming.
thus, finally  <dig>  were used for computational analysis.
ests processing and assembling
we have sequenced  <dig>  cdna clones from macs-separated chicken pgc population cdna library generating  <dig>  sequences.
the est data that are described in this paper have been submitted to the ncbi dbest under accession nos.
dr410159-dr <dig>  assembling and clustering of the est data resulted in  <dig>  unique sequences with  <dig> contigs and  <dig>  singlets.
filtering out possible chimeric sequences with similarity search against chicken genomic sequences were removed  <dig> contigs and  <dig>  singlets resulting in total  <dig>  sequences with  <dig> contigs and  <dig>  singlets.
since the genomic sequences do not have  <dig> % coverage and accuracy, some of the filtered sequences might be genuine sequences but did not correspond with the genomic sequence draft.
average number of ests per contig was about  <dig> .
on the other hands, clustering and assembling of embryonic gonad ests  were resulted in total  <dig>  unique sequences;  <dig> contigs and  <dig>  singlets, respectively.
average est numbers per a contig were approximately  <dig>  in assemblies of embryonic gonad ests.
the unique sequences of the two different sets described in this paper are available at chicken primordial germ cell ests  <cit> .
gene ontology annotation and putative novel transcripts
the number of putative novel transcripts obtained by comparing with the gggi  was total  <dig>  sequences with  <dig> contigs and  <dig>  singlets.
as it was not a cross-species sequence comparison, strict criteria as described in the method were applied to the cut-off values.
for the same reason, identifying the putative novel transcripts can be seen as non-strict criteria.
the reason for applying the strict threshold was to avoid blast hit with paralogous sequences.
functional prediction and gene ontology distribution on the basis of sequence similarity search against non-redundant protein database of the ncbi are shown in additional file  <dig> and additional file 2a.
interestingly, pearson chi-square test of independence of 2nd level go terms between pgcs and novel transcripts data set indicates that a large portion of cellular component unknown term in cellular components would be involved in gene expression of pgcs .
in addition, there were higher portion of behavior and development in biological process, and obsolete molecular function and transporter activity in the novel transcripts than those of pgc set.
digital gene expression profiling
while the comparison of the pearson chi-square test of particular go terms is based on non-redundant data sets, digital gene expression profiling is based upon quantitative differences between two different datasets.
using the audic's test  <cit> , digital gene expression profiling is showed in table  <dig>  there were  <dig> genes expressed significantly higher number of transcripts in pgcs compared with embryonic gonads set.
on the contrary,  <dig> genes in embryonic gonads were up-regulated higher than those in pgc set .
nadh dehydrogenase subunit  <dig> gene, one of two pgc-highly expressed genes, was located in chicken mitochondrial genome and its product was partially related to sperm activity regulated by mitochondrial functions  <cit> , but 40s ribosomal protein sa was not reported in germ cell expression yet.
interestingly, 40s ribosomal protein sa or 37lrp/p <dig> was closely associated to invasive and metastatic activity in cancer cells  <cit> .
during the formation of the undifferentiated gonads, pgcs actively penetrate into gonadal epithelium through blood vessel.
even after settled down in embryonic gonads, they invade from cortical layer into medullar tissue by active migration activity  <cit> .
thus, it might be assumed that 40s ribosomal protein sa is involved in active pgc mobility.
however, further study should be conducted to elucidate its function in pgcs.
among  <dig> gonad-highly expressed genes, several genes such as calmodulin, eukaryotic translation elongation factor  <dig> alpha, ribosomal proteins, thioredoxin, ras homolog, transforming growth factor beta, and vimentin were previously characterize the expression patterns in embryonic gonads as a supportive environmental niche for pgc in other species.
abdallah et al.
<cit>  reported that the somatic form of eukaryotic translation elongation factor  <dig> alpha  mrna is virtually undetectable in male and female germ cells of the adult gonad but is very abundant in embryonic cells after the neurula stage in xenopus laevis.
moreover, the translation pattern of the ef- <dig> alpha is coordinated translational regulation with ribosomal proteins in xenopus laevis during embryogenesis  <cit> .
it is very reasonable result of the up-regulation of antioxidant proteins, thioredoxin, in embryonic gonads because embryonic gonad is more susceptible to oxidant induced damage than adult organs.
li et al.
<cit>  showed that in drosophila, the receptor tyrosine kinase torso activates both stat and ras during the early phase of pgc development, and co-activation of stat and ras is required for pgc proliferation and invasive migration.
members of the transforming growth factor  beta family are pleiotropic cytokines with key roles in tissue morphogenesis and growth and potential roles for tgf beta have been identified in gonad and secondary sex organ development, spermatogenesis and ovarian function  <cit> .
vimentin has been known as an immunohistological marker of sertoli cells which shows over expression during embryonic stage  <cit> .
in mammalian species, ca2+-binding protein, calmodulin was well-known as an activator of fertilized embryo and also was closely related to regulation of interaction between germ cells and neighboring environments at embryonic stages as well as at sexual maturity  <cit> .
we also conducted the alternative serial experiment for gene expression profiling using the massively parallel signature sequencing  from pgcs and embryonic gonads .
using a fdr cut-off of  <dig> , we found  <dig>  and  <dig>  signatures were significantly up-regulated in the pgcs and gonad sample, respectively .
exact binomial probabilities for the situations of n out of n genes were calculated in each sample using the standard binomial formula, where the n is the number of genes identified using ests in each sample, and the probability that the differentially expressed gene of ests will exist in the same sample type of the mpss is the proportion of the des among total mpss in each sample.
interestingly, the up-regulated signatures in the pgcs contained the two genes identified using ests in the pgcs.
the exact binomial probability of exactly  <dig> out of  <dig> was  <dig> e- <dig> with given des proportion in the pgcs .
the up-regulated signatures in the embryonic gonads also contained all of the  <dig> genes identified using ests in the embryonic gonads.
the exact binomial probability of exactly  <dig> out of  <dig> was  <dig> e- <dig> with given des proportion in the gonads .
thus, although the differentially expressed genes identified with ests data was relatively small number, the binomial probabilities indicated that the mpss result was very consistent with the result from the ests data.
CONCLUSIONS
in this study, we could characterize expression gene profiling and identify the significant transcripts expressed in chicken primordial germ cells  as well as embryonic gonads at  <dig>  days.
in the near future, serial experiments will be needed to evaluate biological function and to elucidate interaction in germ cells or a supportive stroma cells during the early embryo development.
germ cell is not only a unique and important cell type compared to other tissues, but also the only cell type that can penetrate from one generation to next generation.
furthermore, at the early embryonic developmental stages, the onset of proliferation and differentiation, germ cell is very tightly regulated by triggering or suppressing the essential genes.
however, collection of germ cells from embryonic stages in aves is very difficulty and complicated and so there are few reports on gene transcript profiling in germ cells retrieved from the embryos to date.
thus, the results in this study would be contributed to investigating the reciprocal interaction between genes during germ cell proliferation and differentiation, and accelerating novel gene mining in germ cells.

 primordial germ cells, the precursor of gametes, have a unique migration activity in birds as well as in mammals. they temporally reside in the extra-embryonic tissue and localize into embryonic gonads. pgcs in mammals are originally derived from the epiblast of the gastrulating embryo and move into embryonic gonads through hindgut by amoeboid movement. in contrast, in birds, pgcs firstly appear from the epiblast in the blastoderm and translocate to the hypoblast of the area pellucida. during the gastrulation, they circulate through the vascular system and shuttle down into the gonadal anlagen. thus, avian pgcs can be collected from germinal crescent or blood vessel, and embryonic gonads. recently, this unique migration pattern of avian pgcs allowed producing germline chimeras by re-transplantation of the pgcs into the blood vessel of recipient embryos. morphological and physiological features of avian germ cells including pgcs have been well characterized and utilized for further studies. however, there are only a few reports on expressed sequence tag analysis and functional genomic study for avian germ cells, especially pgcs in the early embryonic developmental stages. pgc is an important cell type, in which either gene expression or suppression should be regulated temporally and spatially during embryonic developments. according to gene expression switching triggered by interactions with environmental niche, pgcs could maintain their pluripotency or differentiate into germ cells. however, due to technical difficulties, no further progress has been made in functional genomic study and massive novel gene mining in avian pgcs as well as neighboring stroma cells. therefore, this study was conducted to investigate a large-scale est analysis in chicken macs-separated pgcs and compared the expression of the pgc ests with that of embryonic gonad.  retrieval of chicken pgcs by macs treatment embryonic gonads were retrieved from total white leghorn embryos at days of incubation and then the retrieved gonadal cells were treated with magnetic activated cell sorter for separation of pgcs in total embryonic gonadal cells. pgc population ratio after macs separation increased folds than that before macs. thus, we collected × pgcs from embryos for total rna preparation and cdna library construction. the population ratio of chicken pgcs in embryonic gonads was approximately %. so, it is difficulty and complicated to retrieve a large number of chicken pgcs at the early embryonic stages. thus, in this study, gene expression profiling of chicken pgcs was conducted and analyzed with macs-separated chicken pgcs. the morphological and physiological properties of pgcs were unchanged even after macs separation. macs-separated pgcs has the reactivity to germ cell-specific antibodies and the migration capacity into embryonic gonads after re-transplantation into the recipient embryos. cdna library construction from chicken pgcs and est sequencing after in vivo excision with e. coli strain solr, insert sizes of the cdna libraries from pgcs were analyzed by pcr and insert fragments ranged from to kb. titer of primarily cdna library was approximately × pfu/ml on average. subsequently, we massively sequenced ests from pgc cdna library and total ests were sequenced. of ests, sequences were excluded due to low sequencing quality and after vector sequence trimming. thus, finally were used for computational analysis. ests processing and assembling we have sequenced cdna clones from macs-separated chicken pgc population cdna library generating sequences. the est data that are described in this paper have been submitted to the ncbi dbest under accession nos. dr410159-dr assembling and clustering of the est data resulted in unique sequences with contigs and singlets. filtering out possible chimeric sequences with similarity search against chicken genomic sequences were removed contigs and singlets resulting in total sequences with contigs and singlets. since the genomic sequences do not have % coverage and accuracy, some of the filtered sequences might be genuine sequences but did not correspond with the genomic sequence draft. average number of ests per contig was about. on the other hands, clustering and assembling of embryonic gonad ests were resulted in total unique sequences; contigs and singlets, respectively. average est numbers per a contig were approximately in assemblies of embryonic gonad ests. the unique sequences of the two different sets described in this paper are available at chicken primordial germ cell ests. gene ontology annotation and putative novel transcripts the number of putative novel transcripts obtained by comparing with the gggi was total sequences with contigs and singlets. as it was not a cross-species sequence comparison, strict criteria as described in the method were applied to the cut-off values. for the same reason, identifying the putative novel transcripts can be seen as non-strict criteria. the reason for applying the strict threshold was to avoid blast hit with paralogous sequences. functional prediction and gene ontology distribution on the basis of sequence similarity search against non-redundant protein database of the ncbi are shown in additional file and additional file 2a. interestingly, pearson chi-square test of independence of 2nd level go terms between pgcs and novel transcripts data set indicates that a large portion of cellular component unknown term in cellular components would be involved in gene expression of pgcs. in addition, there were higher portion of behavior and development in biological process, and obsolete molecular function and transporter activity in the novel transcripts than those of pgc set. digital gene expression profiling while the comparison of the pearson chi-square test of particular go terms is based on non-redundant data sets, digital gene expression profiling is based upon quantitative differences between two different datasets. using the audic's test, digital gene expression profiling is showed in table there were genes expressed significantly higher number of transcripts in pgcs compared with embryonic gonads set. on the contrary, genes in embryonic gonads were up-regulated higher than those in pgc set. nadh dehydrogenase subunit gene, one of two pgc-highly expressed genes, was located in chicken mitochondrial genome and its product was partially related to sperm activity regulated by mitochondrial functions, but 40s ribosomal protein sa was not reported in germ cell expression yet. interestingly, 40s ribosomal protein sa or 37lrp/p was closely associated to invasive and metastatic activity in cancer cells. during the formation of the undifferentiated gonads, pgcs actively penetrate into gonadal epithelium through blood vessel. even after settled down in embryonic gonads, they invade from cortical layer into medullar tissue by active migration activity. thus, it might be assumed that 40s ribosomal protein sa is involved in active pgc mobility. however, further study should be conducted to elucidate its function in pgcs. among gonad-highly expressed genes, several genes such as calmodulin, eukaryotic translation elongation factor alpha, ribosomal proteins, thioredoxin, ras homolog, transforming growth factor beta, and vimentin were previously characterize the expression patterns in embryonic gonads as a supportive environmental niche for pgc in other species. abdallah et al. reported that the somatic form of eukaryotic translation elongation factor alpha mrna is virtually undetectable in male and female germ cells of the adult gonad but is very abundant in embryonic cells after the neurula stage in xenopus laevis. moreover, the translation pattern of the ef- alpha is coordinated translational regulation with ribosomal proteins in xenopus laevis during embryogenesis. it is very reasonable result of the up-regulation of antioxidant proteins, thioredoxin, in embryonic gonads because embryonic gonad is more susceptible to oxidant induced damage than adult organs. li et al. showed that in drosophila, the receptor tyrosine kinase torso activates both stat and ras during the early phase of pgc development, and co-activation of stat and ras is required for pgc proliferation and invasive migration. members of the transforming growth factor beta family are pleiotropic cytokines with key roles in tissue morphogenesis and growth and potential roles for tgf beta have been identified in gonad and secondary sex organ development, spermatogenesis and ovarian function. vimentin has been known as an immunohistological marker of sertoli cells which shows over expression during embryonic stage. in mammalian species, ca2+-binding protein, calmodulin was well-known as an activator of fertilized embryo and also was closely related to regulation of interaction between germ cells and neighboring environments at embryonic stages as well as at sexual maturity. we also conducted the alternative serial experiment for gene expression profiling using the massively parallel signature sequencing from pgcs and embryonic gonads. using a fdr cut-off of, we found and signatures were significantly up-regulated in the pgcs and gonad sample, respectively. exact binomial probabilities for the situations of n out of n genes were calculated in each sample using the standard binomial formula, where the n is the number of genes identified using ests in each sample, and the probability that the differentially expressed gene of ests will exist in the same sample type of the mpss is the proportion of the des among total mpss in each sample. interestingly, the up-regulated signatures in the pgcs contained the two genes identified using ests in the pgcs. the exact binomial probability of exactly out of was e- with given des proportion in the pgcs. the up-regulated signatures in the embryonic gonads also contained all of the genes identified using ests in the embryonic gonads. the exact binomial probability of exactly out of was e- with given des proportion in the gonads. thus, although the differentially expressed genes identified with ests data was relatively small number, the binomial probabilities indicated that the mpss result was very consistent with the result from the ests data.  in this study, we could characterize expression gene profiling and identify the significant transcripts expressed in chicken primordial germ cells as well as embryonic gonads at days. in the near future, serial experiments will be needed to evaluate biological function and to elucidate interaction in germ cells or a supportive stroma cells during the early embryo development. germ cell is not only a unique and important cell type compared to other tissues, but also the only cell type that can penetrate from one generation to next generation. furthermore, at the early embryonic developmental stages, the onset of proliferation and differentiation, germ cell is very tightly regulated by triggering or suppressing the essential genes. however, collection of germ cells from embryonic stages in aves is very difficulty and complicated and so there are few reports on gene transcript profiling in germ cells retrieved from the embryos to date. thus, the results in this study would be contributed to investigating the reciprocal interaction between genes during germ cell proliferation and differentiation, and accelerating novel gene mining in germ cells. 
after chimeric and problematic sequences were filtered out using the chicken genomic sequences, there were resulting unique sequences consisting of contigs and singlets. however, digital gene expression profiling using the audic's test showed that there were genes expressed significantly with higher number of transcripts in pgcs compared with the embryonic gonads set. on the other hand, genes in embryonic gonads were up-regulated higher than those in the pgc set. this study was conducted to investigate a large-scale expressed sequence tag analysis in chicken pgcs and compare the expression of the pgc ests with that of embryonic gonad. pearson chi-square tests of gene ontology terms in the 2nd level between pgc and embryonic gonad set showed no significance. germ cells are the only cell type that can penetrate from one generation to next generation. at the early embryonic developmental stages, germ cells originally stem from primordial germ cells, and finally differentiate into functional gametes, sperm in male or oocyte in female, after sexual maturity. our results in this study contribute to knowledge of mining novel transcripts and genes involved in germline cell proliferation and differentiation at the early embryonic stages.
 germ cells are the only cell type that can penetrate from one generation to next generation. at the early embryonic developmental stages, germ cells originally stem from primordial germ cells, and finally differentiate into functional gametes, sperm in male or oocyte in female, after sexual maturity. this study was conducted to investigate a large-scale expressed sequence tag analysis in chicken pgcs and compare the expression of the pgc ests with that of embryonic gonad.  we constructed ests from a chicken cdna library of a collection of highly separated embryonic pgcs. after chimeric and problematic sequences were filtered out using the chicken genomic sequences, there were resulting unique sequences consisting of contigs and singlets. pearson chi-square tests of gene ontology terms in the 2nd level between pgc and embryonic gonad set showed no significance. however, digital gene expression profiling using the audic's test showed that there were genes expressed significantly with higher number of transcripts in pgcs compared with the embryonic gonads set. on the other hand, genes in embryonic gonads were up-regulated higher than those in the pgc set.  our results in this study contribute to knowledge of mining novel transcripts and genes involved in germline cell proliferation and differentiation at the early embryonic stages. 