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RESEARCH ARTICLE

Chicken functional genomics: an overview

R. J. Moore A B , T. J. Doran A , T. G. Wise A , S. Riddell A , K. Granger A , T. M. Crowley A , K. A. Jenkins A , A. J. Karpala A , A. G. D. Bean A and J. W. Lowenthal A
+ Author Affiliations
- Author Affiliations

A CSIRO Livestock Industries, Australian Animal Health Laboratory, Private Bag 24, Geelong, Vic. 3220, Australia.

B Corresponding author. Email: rob.moore@csiro.au

Australian Journal of Experimental Agriculture 45(8) 749-756 https://doi.org/10.1071/EA05070
Submitted: 14 February 2005  Accepted: 11 May 2005   Published: 26 August 2005

Abstract

Chickens have undergone intensive selection to produce highly productive strains with excellent growth rates and feed conversion ratios. There does not appear to be any reduction in the rate of strain improvement. The recently completed chicken genome sequencing project and adjunct projects cataloging single nucleotide polymorphisms demonstrate that there is still a high level of genetic variation present in modern breeds. The information provided by genome and transcriptome studies furnishes the chicken biologist with powerful tools for the functional analysis of gene networks. Gene microarrays have been constructed and used to investigate gene expression patterns associated with certain production traits and changes in expression induced by pathogen challenge. Such studies have the potential to identify important genes involved in biological processes influencing animal productivity and health. Fundamental regulatory mechanisms controlled by non-coding RNAs, such as microRNAs, can now be studied following the identification of many potential genes by homology with previously identified genes from other organisms. We demonstrate here that microarrays and northern blotting can be used to detect expression of microRNAs in chicken tissue. Other tools are being used for functional genomic analysis including the production of transgenic birds, still a difficult process, and the use of gene silencing. Gene silencing via RNA interference is having a large impact in many areas of functional genomics and we and others have shown that the mechanisms needed for its action are functional in chickens. The chicken genome sequence has revealed a large number of immune related genes that had not previously been identified in chickens. Functional analysis of these genes is likely to lead to applications aimed at improving chicken health and productivity.

Additional keywords: RNA interference, microRNA, immunogenomics, toll-like receptor.


Acknowledgments

This work is supported in part by the Cooperative Research Centre for the Australian Poultry Industries, the Australian Rural Industry Research and Development Corporation through its Chicken Meat Program and the Australian Egg Corporation Ltd.


References


Abdrakhmanov I, Lodygin D, Geroth P, Arakawa H, Law A, Plachy J, Korn B, Buerstedde J-M (2000) A large database of chicken bursal ESTs as a resource for the analysis of vertebrate gene function. Genome Research 10, 2062–2069.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Ambros V (2004) The functions of animal microRNAs. Nature 431, 350–355.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, Ahringer J, Ruvkun G (2003) Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421, 268–272.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Axelsson E, Webster MT, Smith NGC, Burt DW, Ellegren H (2005) Comparison of the chicken and turkey genomes reveals a higher rate of nucleotide divergence on microchromosomes than macrochromosomes. Genome Research 15, 120–125.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Barstead R (2001) Genome-wide RNAi. Current Opinions in Chemistry and Biology 5, 63–66.
Crossref | GoogleScholarGoogle Scholar | open url image1

Boardman PE, Sanz-Ezquerro J, Overton IM, Burt DW, Bosch E, Fong WT, Tickle C, Brown WRA, Wilson SA, Hubbard SJ (2002) A comprehensive collection of chicken cDNAs. Current Biology 12, 1965–1969.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Bosher JM, Labouesse M (2000) RNA interference: genetic wand and genetic watchdog. Nature Cell Biology 2, E31–E36.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Bourikas D, Stoeckli ET (2003) New tools for gene manipulation in chicken embryos. Oligonucleotides 13, 411–419.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Bourque G, Zdobnov EM, Bork P, Pevzner PA, Tesler G (2005) Comparative architectures of mammalian and chicken genomes reveal highly variable rates of genomic rearrangements across different lineages. Genome Research 15, 98–110.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Brown W, Hubbard SJ, Tickle CA, Wilson SW (2003) The chicken as a model for large-scale analysis of vertebrate gene function. Nature Reviews. Genetics 4, 87–89.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells Science 296, 550–553.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Burgess SC (2004) Proteomics in the chicken: tools for understanding immune responses to avian diseases. Poultry Science 83, 522–573. open url image1

Burnside J, Neiman P, Tang J, Basom R, Talbot R, Aronszajn M, Burt D, Delrow J (2005) Development of a cDNA array for chicken gene expression analysis. BMC Genomics 6, 13.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Burt DW (2002) Origin and evolution of avian microchromosomes. Cytogenetic and Genome Research 96, 97–112.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Burt DW (2004) The chicken genome and the developmental biologist Mechanisms of Development 121, 1129–1135.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Burt DW, Hocking PM (2002) Mapping quantitative trait loci and identification of genes that control fatness in poultry. The Proceedings of the Nutrition Society 61, 441–446.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Carlborg Ö, Kerje S, Schütz K, Jacobsson L, Jensen P, Andersson L (2003) A global search reveals epistatic interactionbetween QTL for early growth in the chicken. Genome Research 13, 413–421.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Chapman SC, Lawson A, Macarthur WC, Wiese RJ, Loechel RH, Burgos-Trinidad M, Wakefield JK, Ramabhadran R, Mauch TJ, Schoenwolf GC (2005) Ubiquitous GFP expression in transgenic chickens using a lentiviral vector. Development 132, 935–940.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Cogburn LA, Wang X, Carre W, Rejto L, Porter TE, Aggrey SE, Simon J (2003) Systems-wide chicken DNA microarrays, gene expression profiling, and discovery of functional genes. Poultry Science 82, 939–951.
PubMed |
open url image1

Cui J, Sofer L, Cloud SS, Burnside J (2004) Patterns of gene expression in the developing chicken thymus. Developmental Dynamics 229, 480–488.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

de Koning DJ, Windsor D, Hocking PM, Burt DW, Law A, Haley CS, Morris A, Vincent J, Griffin H (2003) Quantitative trait locus detection in commercial broiler lines using candidate regions. Journal of Animal Science 81, 1158–1165.
PubMed |
open url image1

Doherty MK, McClean L, Edwards I, McCormack H, McTeir L, Whitehead C, Gackell SJ, Beynon RJ (2004a) Protein turnover in chicken skeletal muscle: understanding protein dynamics on a proteome-wide scale. British Poultry Science 45, S27–S28.
Crossref | PubMed |
open url image1

Doherty MK, McLean L, Hayter JR, Pratt JM, Robertson DH, El-Shafei A, Gaskell SJ, Beynon RJ (2004b) The proteome of chicken skeletal muscle: changes in soluble protein expression during growth in a layer strain. Proteomics 4, 2082–2093.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Duxbury MS, Whang EE (2004) RNA interference: a practical approach. The Journal of Surgical Research 117, 339–344.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Dykxhoorn DM, Novina CD, Sharp PA (2003) Killing the messenger: short RNAs that silence gene expression. Nature Reviews. Molecular Cell Biology 4, 457–467.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Eguchi G, Okada TS (1973) Differentiation of lens from the progeny of chick retinal pigment cells cultured in vitro: a demonstration of a switch of cell types in clonal cell culture. Proceedings of the National Academy of Sciences of the United States of America 70, 1495–1499.
PubMed |
open url image1

Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Emara MG, Kim H (2003) Genetic markers and their application in poultry breeding. Poultry Science 82, 952–957.
PubMed |
open url image1

Etches RJ, Verrinder Gibbins AM (1997) Strategies for the production of transgenic chicken. Methods in Molecular Biology (Clifton, N.J.) 62, 433–450.
PubMed |
open url image1

Fillon V, Morrison M, Zoorob R, Auffray C, Douaire M, Gellin J, Vignal A (1998) Identification of 16 microchromosomes by molecular markers using two-colour fluorescent in situ hybridization (FISH). Chromosome Research 6, 307–314.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Fire A (1999) RNA-triggered gene silencing. Trends in Genetics 15, 358–363.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Gil J, Esteban M (2000) Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 5, 107–114.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Gong ZY, Ju BS, Wang XK, He JY, Wang HY, Sudha PM, Yan T (2002) Green fluorescent protein expression in germline transmitted zebrafish under a stratified epithelial promoter from keratin 8. Developmental Dynamics 223, 204–215.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Hannon GJ (2002) RNA interference. Nature 418, 244–251.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Hillier LW, Miller W, Birney E, Warren W, Hardison RC , et al. (2004) Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432, 695–716.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Hubbard SJ, Grafham DV, Beattie KJ, Overton IM, McLaren SR , et al. (2005) Transcriptome analysis for the chicken based on 19,626 finished cDNA sequences and 485,337 expressed sequence tags. Genome Research 15, 174–183.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Ikeobi CON, Woolliams JA, Morrice DR, Windsor D, Burt DW, Hocking PM (2002) Quantitative trait loci affecting fatness in the chicken. Animal Genetics 33, 428–435.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Iwasaki A, Medzhitov R (2004) Toll-like receptor control of the adaptive immune responses. Nature Immunology 5, 987–995.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Jalving R, van Slot R, van Oost BA (2004) Chicken single nucleotide polymorphism identification and selection for genetic mapping. Poultry Science 83, 1925–1931.
PubMed |
open url image1

Kaiser MG, Deeb N, Lamont SJ (2002) Microsatellite markers linked to Salmonella enterica serovar enteriditis vaccine response in young F1 broiler-cross chicks. Poultry Science 81, 657–663.
PubMed |
open url image1

Kaiser MG, Lamont SJ (2002) Microsatellites linked to Salmonella enterica serovar enteriditis burden in spleen and cecal content of young F1 broiler-cross chicks. Poultry Science 81, 657–663.
PubMed |
open url image1

Kampa D, Burnside J (2002) Jak3-regulated genes: DNA array analysis of concanavalin A-interleukin-2-activated chicken T cells treated with a specific Jak3 inhibitor. Journal of Interferon & Cytokine Research 22, 975–980.
Crossref | GoogleScholarGoogle Scholar | open url image1

Karaca G, Anobile J, Downs D, Burnside J, Schmidt CJ (2004) Herpesvirus of turkeys: microarray analysis of host gene responses to infection. Virology 318, 102–111.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Keeling L, Andersson L, Schutz KE, Kerje S, Fredriksson R, Carlborg O, Cornwallis CK, Pizzari T, Jensen P (2004) Chicken genomics: feather-pecking and victim pigmentation. Nature 431, 645–646.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Kim DH, Rossi JJ (2003) Coupling RNAi-mediated target downregulation with gene replacement. Antisense & Nucleic Acid Drug Development 13, 151–155.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Kwon MS, Koo BC, Choi BR, Lee HT, Kim YH, Ryu W-S, Shim H, Kim J-H, Kim N-H, Kim T (2004) Development of transgenic chickens expressing enhanced green fluorescent protein. Biochemical and Biophysical Research Communications 320, 442–448.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Link CD (2001) Transgenic invertebrate models of age associated neurodegenerative diseases. Mechanisms of Ageing and Development 122, 1639–1649.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Liu H-C, Cheng HH, Tirunagaru V, Sofer L, Burnside J (2001) A strategy to identify positional candidate genes conferring Marek’s disease resistance by integrating DNA microarrays and genetic mapping. Animal Genetics 32, 351–359.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

McGrew MJ, Sherman A, Ellard FM, Lillico SG, Gilhooley HJ, Kingsman AJ, Mitrophanous KA, Sang H (2004) Efficient production of germline transgenic chickens using lentiviral vectors. EMBO Reports 5, 728–733.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Morgan RW, Sofer L, Anderson AS, Bernberg EL, Cui J, Burnside J (2001) Induction of host gene expression following infection of chicken embryo fibroblasts with oncogenic Marek’s disease virus. Journal of Virology 75, 533–539.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Morisson M, Jiguet-Jiglaire C, Lemiere A, Leroux S, Faraut T, Yerle M, Vignal A (2003) A radiation hybrid panel and its use in developing a gene map of the chicken. British Poultry Science 44, 797–798.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Morisson M, Lemiere A, Bosc S, Galan M, Plisson-Petit F , et al. (2002) ChickRH6: a chicken whole-genome radiation hybrid panel. Genetics, Selection, Evolution. 34, 521–533.
Crossref | GoogleScholarGoogle Scholar | open url image1

Mott IW, Ivarie R (2004) cDNA array analysis of Japanese quail lines divergently selected for four-week body weight. Poultry Science 83, 1524–1529.
PubMed |
open url image1

Mozdziak PE, Petitte JN (2004) Status of transgenic chicken models for developmental biology. Developmental Dynamics 229, 414–421.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Mozdziak PE, Pophal S, Borwornpinyo S, Petitte JN (2003) Transgenic chickens expressing beta-galactosidase hydrolyze lactose in the intestine. The Journal of Nutrition 133, 3076–3079.
PubMed |
open url image1

Munir S, Kapur V (2003) Transcriptional analysis of the response of poultry species to respiratory pathogens. Poultry Science 82, 885–892.
PubMed |
open url image1

Nakahara K, Carthew RW (2004) Expanding roles for miRNAs and siRNAs in cell regulation. Current Opinion in Cell Biology 16, 127–133.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. The Plant Cell 2, 279–289.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y (1997) ‘Green Mouse’ as a source of ubiquitous green cells. FEBS Letters 407, 313–319.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Development 16, 948–958.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Pekarik V, Bourikas D, Miglino N, Joset P, Preiswerk S, Stoeckli ET (2003) Screening for gene function in chicken embryo using RNAi and electroporation. Nature Biotechnology 21, 93–96.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Rapp JC, Harvey AJ, Speksnijder GL, Hu W, Ivarie R (2003) Biologically active interferon alpha-2b produced in the egg white of transgenic hens. Transgenic Research 12, 569–575.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Sato F, Nakagawa T, Ito M, Kitagawa Y, Hattori M (2004) Application of RNA interference to chicken embryos using small interfering RNA. The Journal of Experimental Zoology 301A, 820–827.
Crossref | GoogleScholarGoogle Scholar | open url image1

Schmid M, Nanda I, Guttenbach M, Steinlein C, Hoehn H , et al. (2000) First report on chicken genes and chromosomes. Cytogenetics and Cell Genetics 90, 169–218.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Schütz K, Kerje S, Carlborg Ö, Jacobsson L, Andersson L, Jensen P (2002) Analysis of a red junglefowl × White Leghorn intercross reveals trade-off in resource allocation between behaviour and production traits. Behavior Genetics 32, 423–433.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Sewalem A, Morrice DR, Windsor D, Haley CS, Ikeobi CON, Burt DW, Hocking PM (2002) Mapping of quantitative trait loci for body weight at three, six and nine weeks of age in broiler layer cross. Poultry Science 81, 1775–1781.
PubMed |
open url image1

Sharp PA (1999) RNAi and double-stranded RNA. Genes & Development 13, 139–141.
Crossref | PubMed |
open url image1

Smith J, Bruley CK, Paton IR, Dunn I, Jones CT , et al. (2000) Differences in gene density on the chicken macrochromosomes and microchromosomes: a tool for gene discovery in vertebrate genomes. Animal Genetics 31, 96–103.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Smith J, Paton IR, Bruley CK, Windsor D, Burke D, Ponce de Leon FA, Burt DW (2002) Integration of the physical and genetic maps of the chicken macrochromosomes (Gallus gallus) and orientation of linkage groups. Animal Genetics 31, 20–27.
Crossref | GoogleScholarGoogle Scholar | open url image1

Smith J, Speed D, Law AS, Glass EJ, Burt DW (2004) In-silico identification of chicken immune related genes. Immunogenetics 56, 122–133.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD (1998) How cells respond to interferons. Annual Review of Biochemistry 67, 227–264.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Tatsuda K, Fujinaka K (2001) Genetic mapping of the QTL affecting body weight in chickens using a F2 family. British Poultry Science 42, 333–337.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Tirunagaru VG, Sofer L, Cui J, Burnside J (2000) An expressed sequence tag database of T-cell-enriched activated chicken splenocytes: sequence analysis of 5251 clones. Genomics 66, 144–151.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Tuschl T (2002) Expanding small RNA interference. Nature Biotechnology 20, 446–448.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Vallejo RL, Bacon LD, Liu H-C, Witter RL, Groenen MAM, Hillel J, Cheng HH (1997) Genetic mapping of quantitative trait loci affecting susceptibility to Marek’s disease virus induced tumors in F2 intercross chickens. Genetics 148, 349–360. open url image1

van Hemert S, Ebbelaar BH, Smits MA, Rebel JM (2003) Generation of EST and microarray resources for functional genomic studies on chicken intestinal health. Animal Biotechnology 14, 133–143.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

van Hemert S, Hoekman AJ, Smits MA, Rebel JMJ (2004) Differences in intestinal gene expression profiles in broiler lines varying in susceptibility to malabsorption syndrome. Poultry Science 83, 1675–1682.
PubMed |
open url image1

Vargas AO, Fallon JF (2005) Birds have dinosaur wings: the molecular evidence. Journal of Experimental Zoology. Part B. Molecular and Developmental Evolution 304B, 86–90.
Crossref | GoogleScholarGoogle Scholar | open url image1

Wallis JW, Aerts J, Groenen MAM, Crooijmans RPMA, Layman D , et al. (2004) A physical map of the chicken genome. Nature 432, 761–764.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Wang J, He X, Ruan J, Dai M, Chen J , et al. (2005) ChickVD: a sequence variation database for the chicken genome. Nucleic Acids Research 33, D438–D441.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Williams BR (1997) Role of the double-stranded RNA–activated protein kinase (PKR) in cell regulation. Biochemical Society Transcripts 25, 509–513. open url image1

Wong GK, Liu B, Wang J, Zhang Y, Yang X , et al. (2004) A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms. Nature 432, 717–722.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Xiao Y, Hughes AL, Ando J, Matsuda Y, Cheng J-F, Skinner-Noble D, Zhang G (2004) A genome-wide screen identifies a single β-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins. BMC Genomics 5, 56.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Yonash N, Bacon LD, Witter RL, Cheng HH (1999) High resolution mapping and identification of new quantitative trait loci (QTL) affecting susceptibility to Marek’s disease. Animal Genetics 30, 126–135.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Yu JY, DeRuiter SL, Turner DL (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America 99, 6047–6052.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1

Ziauddin J, Sabatini DM (2001) Microarrays of cells expressing defined cDNAs. Nature 411, 107–110.
Crossref | GoogleScholarGoogle Scholar | PubMed | open url image1