1. HERVs (Human endogenous retroviruses) and LTR (long terminal repeat) - like elements are dispersed over 8% of the whole human genome. There are at least 22 independent HERV families within the human genome, which originated from germ-cell infection by the exogenous retrovirus during primate evolution. Elucidation of expression pattern in HERV elements should provide information about fundamental cellular activities and the pathogenesis of multifactorial diseases such as cancer and autoimmune disease. HERV-W env gene is related to multiple sclerosis, and has potential roles for normal differentiation of human villous cytotrophoblast into syncytiotrophoblast. HERV-W env gene was expressed differentially in human tissues. Especially, it was highly expressed in human placenta. This phenomenon indicates HERV-W env gene have the different roles in each tissues. Here, we applied realtime RT-PCR for detection of its expression in various human tissues. We also analysed such amplification using cancer cells and monkey tissues, and discussed in relation to physiological function. ____________________ Retroelement Retroposon SINE Retrotransposon Retrovirus LINE RNA intermediate - LTR element+ LTR element - env + env - RT + RT gagpolenv LTR Human Alu ORF1ORF2P Poly(A) L1 Full-length HERVs/exogenous retrovirus P LTR ORF2 ORF1 Poly(A) Yeast Ty1/copia/truncated HERVs Human THE1 Pseudogenes LTR provirus LTR Enodgenous retrovirus Exogenous Retroviruses Retrotransposons Endogenous Retroviruses budding proteins (gag, env) translation infection REVERSE TRANSCRIPTION transcription integration cDNA AAA mRNA AAA transcription LTR re - integration translation proteins (gag, env) REVERSE TRANSCRIPTION cDNA Particle formation(budding) LTR re - integration Particle formation no re-infection LTR retrotransposon AAA transcription mRNA cDNA REVERSE TRANSCRIPTION Cellular gene pseudogene splicing AAA REVERSE TRANSCRIPTION cDNA integration transcription spliced- mRNA translation proteins (gag) Other region 16% Gene-related Sequence 36% LINE 20% SINE 13% Coding sequence 3% Pseudogene 1% HERV element 8% DNA element 3% Primer Design Twenty Different Human tissue cDNAs Quantitative Analysis Realtime RT-PCR using SYBR Green I Extension(II) phase Extension(I) phase End of PCR cycle Annealing phase SYBR Green I Detection of fluorescence Experimental Procedure relative quantification normalisation via one reference gene via reference gene index >3HKG external calibration curve without any reference gene without real-time PCR efficiency correction 2 (- △△ CP) with real-time PCR efficiency correction REST, qGene LC software & etc... 1. Gapdh standard 2. W-env standard 3. Meting graph 5. Tissue melting graph 4. Tissue expression graph 6. Relative quantitative analysis of w-env expression 7. Relative quantitative analysis of w-env expression except for placenta Yi JM and Kim HM and Kim HS 2004. Expression of the human endogenous retrovirus HERV-W family in various human tissues and cancer cells. J. Gen. Virol. 85: 1203-1210. Yi JM, Osamu T and Kim HS 2003. Molecular characterization and phylogenetic relationship of HERV-W family in the Macaca fuscata. Arch. Virol. 148(8) 1613-1622. Placenta Adrenal gland Bone marrowCerebellumBrainFetal brain Fetal liver Heart KidneyLiverLungProstateSalivary glandSkeletal muscleSpinal cordTestisThymusThyroidTracheaUterus Adrenal gland Bone marrowCerebellumBrainFetal brain Fetal liver Heart KidneyLiver Lung ProstateSalivary glandSkeletal muscleSpinal cordTestisThymusThyroidTracheaUterus