genomic imprinting , in which the monoallelic expression of certain genes depends on the parent of origin due to epigenetic regulation in somatic cells , is vital for fetal growth and development , function of the placenta , and postnatal behaviour in mammals . many researches have reported that disruption of genomic imprinting is responsible for congenital disorders , cancers , and other types of diseases such as bws ( beckwith - wiedemann syndrome ) and pws ( prader - willi syndrome ) [ 14 ] . currently , about 2114 imprinted transcripts and 600 potentially imprinted genes have been estimated in the genome of mice [ 5 , 6 ] , of which about 160 imprinted genes have been described ( http://igc.otago.ac.nz/home.html ) , but only several genes have been reported to be imprinted in pigs . therefore , it is necessary to characterize more imprinted genes in the pig to offer new data and insights to the comparative genome research as well as porcine functional genomics . most imprinted genes aggregate within the genome and gene order and imprinting in each imprinted domain show considerable similarity between mouse and human [ 7 , 8 ] . it has been reported that cdkn1c and kcnq1ot1 reside in an imprinted cluster on human 11p15.5 , syntenic to mouse distal chromosome 7 . genes in this region include osbpl5 , cars , nap1l4 , phlda2 , slc22a18 , slc22a18as , cdkn1c , kcnq1dn , kcnq1ot1 , kcnq1 , tssc4 , cd81 , and ascl2 . imprinting disruption of the cdkn1c / kcnq1ot1 domain is involved in the development of both bws and cancer . cdkn1c ( cyclin - dependant kinase inhibitor ) and nap1l4 ( nucleosome assembly protein 1-like 4 ) are two of particular interest genes within the cdkn1c / kcnq1ot1 imprinted cluster in human and mouse . the cdkn1c , also known as p57 , encodes a cyclin - dependent kinase inhibitor ( cdki ) that belongs to the cip / kip family , and it is a putative tumor suppressor gene [ 9 , 10 ] . mutations in cdkn1c have been identified in approximately 520% of patients with bws [ 11 , 12 ] , and mice with maternally inherited deletion of cdkn1c showed some features of bws , while in sporadic cancers and embryonal tumors , mutations of cdkn1c are very rare [ 1418 ] . nap1l4 , also called nap-2 , codes for a protein with homology to naps involved in regulating gene expression by delivering histones from the cytoplasm to the chromatin assembly machinery . these results suggest that cdkn1c and nap1l4 are two of the important genes responsible for the development of bws and cancer .   at present , there are a lot of reports about the imprinting status of cdkn1c and nap1l4 both in human and mouse . cdkn1c is expressed from the maternal allele , while nap1l4 is biallelically expressed in all the tissues except maternal expression in mouse placenta . in the pig , cdkn1c is expected of maternal expression . despite of these advances , hence , tissue expression patterns and imprinting status of porcine cdkn1c and nap1l4 were investigated in this work . this study will contribute to increasing the understanding of functional importance of cdkn1c and nap1l4 in pigs . reciprocal crossings were performed using 8 landrace ( ld ) and 8 indigenous china breed rongchang ( rc ) pigs ( 4 boars and 4 sows of each breed ) . tissues including heart , stomach , skeletal muscle , kidney , lung , liver , small intestine , bladder , tongue , spleen , fat , ovary and placenta were collected from 23 f1 pigs , which included 10 neonatal pigs within 24  h after birth ( 5 pigs from three parent pairs of ld  rc and 5 pigs from three parent pairs of rc  ld ) as well as 7 and 6 one - month pigs from four parent pairs of ld  rc and rc  ld , respectively . ear tissues of parental pigs for genomic dna isolation and genotyping of cdkn1c and nap1l4 were also collected . total rna was isolated from frozen tissues using trizol reagent ( invitrogen , san diego , usa ) and treated with dnase i ( takara , japan ) . reverse transcription was carried out using amv reverse transcriptase xl ( takara , japan ) and oligo(dt ) primer at 42c for 50  min , 70c for 15  min . both reactants with and without reverse transcriptase were used as positive and negative samples , respectively , for gene cloning and imprinting analysis . the cdna sequences of human genes cdkn1c ( nm_000076 ) and nap1l4 ( nm_005969 ) were used to search for available expressed sequence tags ( ests ) in the pig ests database using blastn program ( http://www.ncbi.nlm.nih.gov/blast/ ) . the homologous sequences , sharing greater than 80% identity and match of at least 100  bp , were assembled into ests contigs for primer design . primer pair cdf1/r1 was used for cdkn1c , while naf1/r1 for nap1l4 ( table 1 ) . to obtain full - length cdna of the two genes , 5- and 3-race were performed with smart race cdna amplification kit ( clontech , usa ) and the rna la pcr kit ( takara , japan ) , respectively , according to the supplier 's instructions . for 5-race , sequence - specific primers 5cdgsp1 and 5cdgsp2 ( nested ) were designed for cdkn1c , while 5nagsp1 and 5nagsp2 ( nested ) for nap1l4 . for 3-race , thermocycling conditions of pcr reactions were initial denaturation at 94c for 5  min , followed by 35 cycles of denaturation at 94c for 30  s , annealing at 5360c for 30  s ( table 1 ) , and extension at 72c for 30  s , with a final extension at 72c for 10  min . pcr products were then sequenced at commercial service ( invitrogen , shanghai , china ) .   to obtain intron sequence for rh mapping , alignment of porcine cdkn1c and nap1l4 with human corresponding genes ( nm_000076 , nm_005969 ) according to the consensus sequences , primers inf1/r1 and inf2/r2 ( table 1 ) were designed for amplification of cdkn1c intron 2 and nap1l4 intron 3 . primer pairs mf1/r1 and mf2/r2 ( table 1 ) based on the introns above were used for pcr typing of the porcine radiation hybrid ( imprh ) panel , which were kindly provided by inra ( france ) . pcr was performed in duplicate in a total volume of 10  l , containing 25  ng imprh dna samples , 0.3  m of each primer , 75  m of each dntp , 1.5  mm mgcl2 , and 1  u taq dna polymerase ( takara , japan ) . the pcr conditions were as follows : 3  min at 94c , followed by 35 cycles of 30  s at 94c , 30  s at 60c , 30  s at 72c , and an extra 7  min extension at 72c . in addition , pcr reactions were carried out with pig genomic dna ( as positive control ) and without dna ( as negative control ) . the statistical analyses of the pcr results were performed with the imprh mapping tool accessible at http://rhdev.toulouse.inra.fr/. two - point and multiple - point rh analyses were used for the identification of linkage groups using lod score threshold of 5.0 . snps of cdkn1c and nap1l4 genes were searched via alignment of landrace and rongchang cdna sequences . genotyping was performed for single - nucleotide polymorphisms ( snps ) in each gene using genomic dna of the 23 f1 individuals . snps were detected by direct sequencing of pcr products using primer pairs ijf1/r1 , ijf2/r2 ( table 1 ) , for cdkn1c and nap1l4 , respectively . the allelic expression pattern of each gene was analyzed by rt - pcr of total rna from samples heterozygous for snps . pcr products by each of the primer pairs described above were then sequenced in both directions . to eliminate the possibility of genomic dna contamination in the rt - pcr , primers -actinf / r spanning intron 3 of pig -actin were designed based on porcine -actin ( ay550069 ) . quantitative real - time pcr was performed with sybr premix ex taqtm ii kit in a bio - rad iq 5 machine ( usa ) . primers qf1/r1 , qf2/r2 and qf3/r3 ( table 1 ) were used to amplify a 136 , 128 , and 165  bp cdna for cdkn1c , nap1l4 , and rpl32 ( ribosomal protein l32 ) , respectively . total rna extracted from each tissue sample was reverse transcribed using primescript rtase and oligo(dt ) ( takara , japan ) . the reaction volume of 25  l contained 12.5  l sybr premix ex taq ii ( takara , japan ) , 0.4  m each of primers , and 2.5  l template cdna . a standard curve for analysis of the expression data was derived for each single gene from a serial dilution of the pool of cdna samples . the endogenous control gene rpl32 , which showed stable expression in our published data , was used . anova analysis and multiple comparisons were applied to each gene to verify whether the expression difference between tissues or ages was significant . the full - length cdna spanning 1171  bp was assembled for porcine cdkn1c after sequencing the pcr products of primers cdf1/r1 as well as 5-race ( genbank accession no . sequence analysis showed that the gene contained an open reading frame ( orf ) of 786  bp ( from nt 162 to nt 947 of hq679903 ) , encoding a 261-amino acid protein . the orf is 79% identical in amino acid sequence and 85% identical in nucleotide sequence to human sequences ( nm_000076 ) . the full - length cdna of nap1l4 is 2655  bp ( hq679902 ) with 470  bp 5-utr , 1024  bp 3-utr , and 1161  bp orf , which codes for 386 amino acids . the porcine nap1l4 utilizes a polyadenylation site ( attaaa ) , located at position 2617 of hq679902 . the orf is 94% identical in amino acid sequence and 86% identical in nucleotide sequence to human sequences ( nm_005969 ) . in human and mouse , cdkn1c and nap1l4 genes the two - point analysis showed that both cdkn1c ( lod 15.78 , distance 0.15  cr , and retention frequency 16% ) and nap1l4 ( lod 17.94 , distance 0.11  cr , and retention frequency 19% ) were closely linked to imprh06175 , on sus scrofa chromosome 2 ( ssc2 ) . the multiple - point analysis demonstrated that cdkn1c was linked to 311840215 ( upper marker , dist 0.34  cr ) and imprh06175 ( lower marker , dist 0.14  cr ) , while nap1l4 was linked to 311840215 ( upper marker , dist 0.36  cr ) and imprh06175 ( lower marker , dist 0.10  cr ) . the expression patterns of porcine cdkn1c were determined by relative quantitative real - time pcr analysis with total rnas isolated from tissues of 5 neonatal and 7 one - month hybrids of ld  rc . for each age , rna of a tissue was used as a biological repeat . in neonatal pigs , the mrna level of cdkn1c in placenta was amazingly high ( p < 0.01 ) , while it was very low in other tissues and with no differences between them ( figure 1(a ) ) . in one - month - old pigs , highest expression levels were observed in lung and kidney ( p < 0.01 ) , followed by skeletal muscle and heart ( p < 0.01 ) . fewer cdkn1c transcripts were present in spleen , tongue , bladder , stomach , liver , fat , and small intestine ( p < 0.01 ) . interestingly , transcript levels of porcine cdkn1c were upregulated in lung and skeletal muscle , and downregulated in tongue , bladder , and small intestine ( p < 0.01 ) , but no difference was seen in the other tissues after birth . alignment of rongchang and landrace sequences revealed an snp ( a / g ) at position 916 of cdkn1c ( hq679903 ) . since the polymorphism did not provide any available rflp site , direct sequencing of rt - pcr products was used for the discrimination of the expressed alleles . sequencing of the pcr products revealed that three newborn pigs from rc  ld and four from ld  rc occurred to be heterozygous for the snp , while their parental pigs were homozygous for the allele a and g , respectively . the ( rc  ld ) f1 pigs only expressed the landrace allele g , and ( ld  rc ) f1 pigs only expressed the rongchang a allele in placenta ( figure 2(a ) ) as well as in tongue , bladder , ovary , spleen , liver , skeletal muscle , stomach , and small intestine . however , biallelic expression of porcine cdkn1c was detected in lung and kidney ( figure 2(a ) ) . the expression level was significantly higher in placenta ( p < 0.01 ) , followed by lung and skeletal muscle ( p < 0.05 ) . fewer nap1l4 transcripts were present in spleen , tongue , bladder , ovary , liver , stomach and small intestine ( p < 0.05 ) ( figure 1(b ) ) . in contrast , at the age of one month , highest levels of expression were observed in lung , heart , and kidney ( p < 0.01 ) and the lowest expression were detected in spleen and tongue ( p < 0.01 ) . compared with neonatal tissues , expression of porcine nap1l4 was upregulated in heart and kidney , and downregulated in skeletal muscle ( p < 0.01 ) , but no significant changes were seen in the other tissues . a t / c snp was detected between rongchang and landrace pigs at position 2461 of nap1l4 ( hq679902 ) . a total of five dna samples from ( rc  ld ) f1 pigs and four dna samples from ( ld  rc ) f1 pigs were heterozygous for this polymorphism , while their parents landrace and rongchang pigs were c / c and t / t homozygotes , respectively . direct sequencing of rt - pcr products demonstrated biallelic transcription of nap1l4 in lung , heart , kidney , tongue , bladder , ovary , spleen , liver , skeletal muscle , stomach , small intestine , and spleen of newborn pigs . the biallelic expression of porcine nap1l4 in skeletal muscle is illustrated in figure 2(b ) . in this study , cdkn1c and nap1l4 genes were cloned , and their chromosome location , expression patterns , and imprinting were further analyzed for the first time in pigs . we use rongchang and landrace breeds for analysis of allelic expression due to the fact that f1 hybrids of the two breeds are useful for detecting imprinting of genes . sequence analysis showed that porcine cdkn1c and nap1l4 have high homology with those of human in amino acid and in nucleic acid , respectively , and both genes are closely linked in the region between markers imprh06175 and 311840215 on ssc2 , syntenic to mice distal chromosome 7 and human 11p15.5 . cdkn1c is a potent inhibitor of several g1 cyclin / cdk complexes , and its overexpression leads to cell - cycle arrest in the g1 phase [ 26 , 27 ] . the biological functions and distribution of cdkn1c in tissues have been widely investigated . in human and mouse , researches have shown that cdkn1c is mostly expressed in placenta and at low levels in other tissues , while it is expressed in adult heart , brain , lung , skeletal muscle , kidney , and pancreas [ 28 , 29 ] . recently , shukla et al .   revealed that mrna levels of cdkn1c were altered in placenta of intrauterine growth restriction ( iugr ) and alcohol - consuming dams compared to the controls ; thus , they were selected as the biomarkers for iugr and intrauterine alcohol exposure . mutations in mouse cdkn1c result in the appearance of large thrombotic lesions in the labyrinth zone in mouse placenta . our work showed that the mrna abundance of cdkn1c was highest in porcine neonatal placenta , while its high expression levels were detected in lung and kidney of one - month - old pigs . in general , the expression pattern of porcine cdkn1c correlates well with the human and mouse gene , and the high levels in expression of cdkn1c in porcine placenta illustrate its important roles in porcine placental growth and development . nap1l4 has been found to be localized mainly to the cytoplasm and a minor population of them to be in the nucleus , mediating nucleosome formation . hu et al .   found that nap1l4 is ubiquitously expressed , showing a threefold higher level of expression in testis than in other tissues . interestingly , our work also showed that nap1l4 was ubiquitously expressed in porcine tissues , but higher expression of nap1l4 was detected in neonatal placenta than in the other tissues . as nap1l4 mediates angiogenesis , and it is significantly higher expressed in red - feather taiwan country chickens of high egg productions than in the subpopulations of low egg productions . these studies indicate that the functions of nap1l4 contribute to reproduction performance in animals and may play a role in placental functions . mouse nap1l4 is paternally repressed in placenta and biallelically expressed in other embryo tissues , whereas biallelic expression of human nap1l4 can be detected in all tissues and ages tested by higashimoto et al . . like in humans , nap1l4 in pigs was biallelically expressed in all tissues detected in this study . furthermore , our data showed maternal expression of porcine cdkn1c in heart , tongue , bladder , ovary , spleen , liver , skeletal muscle , stomach , small intestine and placenta , consistent with previous data of greater expression of cdkn1c from parthenote than from biparental samples . the biallelic expression of cdkn1c in lung and kidney in the pig differs from monoallelic expression in the same tissues in humans . this is because the degree of imprinting is often tissue and species specific because of different epigenetic modification . in humans and mice , it has been documented that cdkn1c imprinting is regulated by kcnq1ot1 antisense - rna transcript originating from intron 10 of the maternally expressed kcnq1 ( kvlqt1 ) gene . a cpg island located at the promoter of kcnq1ot1 , referred to as kvdmr1 or kcnq1 imprinting control region ( icr ) , is unmethylated on the paternal allele and methylated on the maternal allele , favouring expression of kcnq1ot1 from the paternal allele that leads to the propagation of silencing of neighboring genes including cdkn1c . while the kcnq1 icr has not been characterized in pigs , gene order and gene structure within this cluster are highly conservative between human , mouse , chicken , and tammar . this information lends support to the hypothesis that different epigenetic modification of kcnq1 icr or other mechanisms terminatie the kcnq1ot1 transcripts in porcine lung and kidney . further studies are needed to elucidate the mechanisms associated with biallelic expression of cdkn1c in lung and kidney of pigs . the full - length cdna of porcine nap1l4 and cdna including 5-utr and orf of porcine cdkn1c was cloned in this study . imprinting analysis showed that nap1l4 was biallelically expressed in the tissues tested , while cdkn1c was maternally expressed in tissues except lung and kidney in pigs , confirming the results by bischoff et al .   that cdkn1c is expected of maternal expression . this study revealed a novel imprinted domain in the pig and provided data for further investigation of functional importance of porcine cdkn1c and nap1l4 .
cdkn1c and nap1l4 in human cdkn1c / kcnq1ot1 imprinted domain are two key candidate genes responsible for bws ( beckwith - wiedemann syndrome ) and cancer . in order to increase understanding of these genes in pigs , 
 their cdnas are characterized in this paper . by the imprh panel , porcine cdkn1c and nap1l4 genes 
 were assigned to porcine chromosome 2 , closely linked with imprh06175 and with lod of 15.78 and 17.94 , respectively . by real - time quantitative rt - pcr and polymorphism - based method , tissue and allelic expression of both genes 
 were determined using f1 pigs of rongchang and landrace reciprocal crosses . 
 the transcription levels of porcine cdkn1c and nap1l4 were significantly higher in placenta than in other neonatal tissues ( p < 0.01 ) although both genes showed the highest expression levels in the lung and kidney of one - month pigs ( p < 0.01 ) . 
 imprinting analysis demonstrated that in pigs , cdkn1c was maternally expressed in neonatal heart , tongue , bladder , ovary , spleen , liver , skeletal muscle , stomach , small intestine , and placenta and biallelically expressed in lung and kidney , while nap1l4 was biallelically expressed in the 12 neonatal tissues examined . 
 it is concluded that imprinting of cdkn1c is conservative in mammals but has tissue specificity in pigs , and imprinting of nap1l4 is controversial in mammalian species .