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Designing Transgenes for Optimal Expression

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Advanced Protocols for Animal Transgenesis

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Abstract

In theory, designing a DNA construct to be used for transgene purposes, for standard pronuclear microinjection, would seem a rather easy task. The combination of a given promoter and some regulatory elements of choice, driving the expression of the construct to the desired tissue, with a suitable coding region of the gene of interest, and finishing the construct with an adequate transcription terminator would appear to be a straightforward process. However, chromosomal position effects, variegated expression, non-expressing transgenic mouse lines or those displaying ectopic and unexpected patterns of transgene expression are not uncommon. Therefore, great care should be invested in the design of the transgene, with optimal transgene expression the goal. With very few exceptions, there is no reliable catalogue of plasmid-based promoters that one could refer to when looking for robust tissue-specific transgene expression. Instead, BAC- and YAC-based transgenes have proven to produce optimal results, thus suggesting that genomic-type constructs may be more reliable as promoters than standard plasmid-type constructs. This and other observations will be discussed in this chapter. Three golden rules must be applied when designing a transgene (1) transgenes should not contain vector sequences; (2) transgenes should not contain DNA sequences derived from prokaryotic genomes; and, most importantly, (3) the more a transgene resembles the corresponding endogenous locus, the better it will behave in terms of expression levels and pattern. These very basic rules should be taken into account when preparing a DNA construct to be used as transgene, enabling easy removal of vector and prokaryotic sequences that are no longer required (and will normally have a detrimental effect upon transgene expression) and allowing the inclusion of genomic sequences that are fundamental for the faithful regulation of the locus.

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References

  1. Chowdhury K, Bonaldo P, Torres M, Stoykova A, Gruss P (1997) Evidence for the stochastic integration of gene trap vectors into the mouse germline. Nucleic Acids Res 25:1531–1536

    Article  PubMed  CAS  Google Scholar 

  2. Kitajima K, Takeuchi T (1998) Mouse gene trap approach: identification of novel genes and characterization of their biological functions. Biochem Cell Biol 76:1029–1037

    Article  PubMed  CAS  Google Scholar 

  3. Stanford WL, Cohn JB, Cordes SP (2001) Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet 2:756–768

    Article  PubMed  CAS  Google Scholar 

  4. Boshart M, Klüppel M, Schmidt A, Schütz G, Luckow B (1992) Reporter constructs with low background activity utilizing the cat gene. Gene 110:129–130

    Article  PubMed  CAS  Google Scholar 

  5. Beermann F, Ruppert S, Hummler E, Bosch FX, Müller G, Rüther U, Schütz G (1990) Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice. EMBO J 9:2819–2826

    PubMed  CAS  Google Scholar 

  6. Montoliu L, Blendy JA, Cole TJ, Schütz G (1995) Analysis of perinatal gene expression: hormone response elements mediate activation of a lacZ reporter gene in liver of transgenic mice. Proc Natl Acad Sci USA 92:4244–4248

    Article  PubMed  CAS  Google Scholar 

  7. Giraldo P, Martínez A, Regales L, Lavado A, García-Díaz A, Alonso A, Busturia A, Montoliu L (2003) Functional dissection of the mouse tyrosinase locus control region identifies a new putative boundary activity. Nucleic Acids Res 31:6290–6305

    Article  PubMed  CAS  Google Scholar 

  8. Lavado A, Jeffery G, Tovar V, de la Villa P, Montoliu L (2006) Ectopic expression of tyrosine hydroxylase in the pigmented epithelium rescues the retinal abnormalities and visual function common in albinos in the absence of melanin. J Neurochem 96:1201–1211

    Article  PubMed  CAS  Google Scholar 

  9. Overbeek PA, Aguilar-Cordova E, Hanten G, Schaffner DL, Patel P, Lebovitz RM, Lieberman MW (1991) Coinjection strategy for visual identification of transgenic mice. Transgenic Res 1:31–37

    Article  PubMed  CAS  Google Scholar 

  10. Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM (1982) Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300:611–615

    Article  PubMed  CAS  Google Scholar 

  11. Giraldo P, Montoliu L (2001) Size matters: use of YACs. BACs and PACs in transgenic animals. Transgenic Res 10:83–103

    Article  PubMed  CAS  Google Scholar 

  12. Zhang Q, Triplett AA, Harms DW, Lin WC, Creamer BA, Rizzino A, Wagner KU (2010) Temporally and spatially controlled expression of transgenes in embryonic and adult tissues. Transgenic Res 19:499–509

    Article  PubMed  CAS  Google Scholar 

  13. Hammer RE, Swift GH, Ornitz DM, Quaife CJ, Palmiter RD, Brinster RL, MacDonald RJ (1987) The rat elastase I regulatory element is an enhancer that directs correct cell specificity and developmental onset of expression in transgenic mice. Mol Cell Biol 7:2956–2967

    PubMed  CAS  Google Scholar 

  14. Kollias G, Hurst J, deBoer E, Grosveld F (1987) The human beta-globin gene contains a downstream developmental specific enhancer. Nucleic Acids Res 15:5739–5747

    Article  PubMed  CAS  Google Scholar 

  15. Aronow B, Lattier D, Silbiger R, Dusing M, Hutton J, Jones G, Stock J, McNeish J, Potter S, Witte D, Wiginton D (1989) Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene. Genes Dev 3:1384–1400

    Article  PubMed  CAS  Google Scholar 

  16. Brooks AR, Nagy BP, Taylor S, Simonet WS, Taylor JM, Levy-Wilson B (1994) Sequences containing the second-intron enhancer are essential for transcription of the human apolipoprotein B gene in the livers of transgenic mice. Mol Cell Biol 14:2243–2256

    Article  PubMed  CAS  Google Scholar 

  17. Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD (1988) Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci USA 85:836–840

    Article  PubMed  CAS  Google Scholar 

  18. Palmiter RD, Sandgren EP, Avarbock MR, Allen DD, Brinster RL (1991) Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci USA 88:478–482

    Article  PubMed  CAS  Google Scholar 

  19. Whitelaw CB, Archibald AL, Harris S, McClenaghan M, Simons JP, Clark AJ (1991) Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice. Transgenic Res 1:3–13

    Article  PubMed  CAS  Google Scholar 

  20. Petitclerc D, Attal J, Théron MC, Bearzotti M, Bolifraud P, Kann G, Stinnakre MG, Pointu H, Puissant C, Houdebine LM (1995) The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice. J Biotechnol 40:169–178

    Article  PubMed  CAS  Google Scholar 

  21. Schedl A, Montoliu L, Kelsey G, Schütz G (1993) A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice. Nature 362:258–261

    Article  PubMed  CAS  Google Scholar 

  22. Houdebine LM (2000) Transgenic animal bioreactors. Transgenic Res 9:305–320

    Article  PubMed  CAS  Google Scholar 

  23. Houdebine LM (2002) Animal transgenesis: recent data and perspectives. Biochimie 84:1137–1141

    Article  PubMed  CAS  Google Scholar 

  24. Houdebine LM (2007) Transgenic animal models in biomedical research. Methods Mol Biol 360:163–202

    PubMed  CAS  Google Scholar 

  25. Yull F, Binas B, Harold G, Wallace R, Clark AJ (1997) Transgene rescue in the mammary gland is associated with transcription but does not require translation of BLG transgenes. Transgenic Res 6:11–17

    Article  PubMed  CAS  Google Scholar 

  26. Palmiter RD, Brinster RL (1986) Germ-line transformation of mice. Annu Rev Genet 20:465–499

    Article  PubMed  CAS  Google Scholar 

  27. Giménez E, Lavado A, Giraldo P, Montoliu L (2003) Tyrosinase gene expression is not detected in mouse brain outside the retinal pigment epithelium cells. Eur J Neurosci 18:2673–2676

    Article  PubMed  Google Scholar 

  28. Montoliu L, Larue L, Beermann F (2004) On the use of regulatory regions from pigmentary genes to drive the expression of transgenes in mice. Pigment Cell Res 17:188–190

    Article  PubMed  CAS  Google Scholar 

  29. Montoliu L, Chávez S, Vidal M (2000) Variegation associated with lacZ in transgenic animals: a warning note. Transgenic Res 9:237–239

    Article  PubMed  CAS  Google Scholar 

  30. Ramírez A, Milot E, Ponsa I, Marcos-Gutiérrez C, Page A, Santos M, Jorcano J, Vidal M (2001) Sequence and chromosomal context effects on variegated expression of keratin 5/lacZ constructs in stratified epithelia of transgenic mice. Genetics 158:341–350

    PubMed  Google Scholar 

  31. Kioussis D, Festenstein R (1997) Locus control regions: overcoming heterochromatin-induced gene inactivation in mammals. Curr Opin Genet Dev 7:614–619

    Article  PubMed  CAS  Google Scholar 

  32. Giménez E, Giraldo P, Jeffery G, Montoliu L (2001) Variegated expression and delayed retinal pigmentation during development in transgenic mice with a deletion in the locus control region of the tyrosinase gene. Genesis 30:21–25

    Article  PubMed  Google Scholar 

  33. Giménez E, Lavado A, Jeffery G, Montoliu L (2005) Regional abnormalities in retinal development are associated with local ocular hypopigmentation. J Comp Neurol 485:338–347

    Article  PubMed  Google Scholar 

  34. Wilkie TM, Brinster RL, Palmiter RD (1986) Germline and somatic mosaicism in transgenic mice. Dev Biol 118:9–18

    Article  PubMed  CAS  Google Scholar 

  35. Whitelaw CB, Springbett AJ, Webster J, Clark J (1993) The majority of G0 transgenic mice are derived from mosaic embryos. Transgenic Res 2(1):29–32

    Article  PubMed  CAS  Google Scholar 

  36. Pravtcheva DD, Wise TL, Ensor NJ, Ruddle FH (1994) Mosaic expression of an Hprt transgene integrated in a region of Y heterochromatin. J Exp Zool 268:452–468

    Article  PubMed  CAS  Google Scholar 

  37. Koetsier PA, Mangel L, Schmitz B, Doerfler W (1996) Stability of transgene methylation patterns in mice: position effects, strain specificity and cellular mosaicism. Transgenic Res 5:235–244

    Article  PubMed  CAS  Google Scholar 

  38. Wilkins AS (1990) Position effects, methylation and inherited epigenetic states. Bioessays 12:385–386

    Article  PubMed  CAS  Google Scholar 

  39. Grosveld F, de Boer E, Dillon N, Gribnau J, McMorrow T, Milot E, Trimborn T, Wijgerde M, Fraser P (1998) The dynamics of globin gene expression and position effects. Novartis Found Symp 214:67–79, discussion 79–86

    PubMed  CAS  Google Scholar 

  40. Grosveld F, van Assendelft GB, Greaves DR, Kollias G (1987) Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell 51:975–985

    Article  PubMed  CAS  Google Scholar 

  41. Lang G, Mamalaki C, Greenberg D, Yannoutsos N, Kioussis D (1991) Deletion analysis of the human CD2 gene locus control region in transgenic mice. Nucleic Acids Res 19:5851–5856

    Article  PubMed  CAS  Google Scholar 

  42. Ganss R, Montoliu L, Monaghan AP, Schütz G (1994) A cell-specific enhancer far upstream of the mouse tyrosinase gene confers high level and copy number-related expression in transgenic mice. EMBO J 13:3083–3093

    PubMed  CAS  Google Scholar 

  43. Montoliu L, Umland T, Schütz G (1996) A locus control region at −12 kb of the tyrosinase gene. EMBO J 15:6026–6034

    PubMed  CAS  Google Scholar 

  44. Grosveld F, Antoniou M, Berry M, De Boer E, Dillon N, Ellis J, Fraser P, Hanscombe O, Hurst J, Imam A, Lindenbaum M, Philipsen S, Pruzina S, Strouboulis J, Raguz-Bolognesi S, Talbot D (1993) The regulation of human globin gene switching. Philos Trans R Soc Lond B Biol Sci 339:183–191

    Article  PubMed  CAS  Google Scholar 

  45. Bonifer C, Vidal M, Grosveld F, Sippel AE (1990) Tissue specific and position independent expression of the complete gene domain for chicken lysozyme in transgenic mice. EMBO J 9:2843–2848

    PubMed  CAS  Google Scholar 

  46. McKnight RA, Shamay A, Sankaran L, Wall RJ, Hennighausen L (1992) Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc Natl Acad Sci USA 89:6943–6947

    Article  PubMed  CAS  Google Scholar 

  47. McKnight RA, Spencer M, Wall RJ, Hennighausen L (1996) Severe position effects imposed on a 1 kb mouse whey acidic protein gene promoter are overcome by heterologous matrix attachment regions. Mol Reprod Dev 44:179–184

    Article  PubMed  CAS  Google Scholar 

  48. Gutiérrez-Adán A, Pintado B (2000) Effect of flanking matrix attachment regions on the expression of microinjected transgenes during preimplantation development of mouse embryos. Transgenic Res 9:81–89

    Article  PubMed  Google Scholar 

  49. Moreira PN, Pérez-Crespo M, Ramírez MA, Pozueta J, Montoliu L, Gutiérrez-Adán A (2007) Effect of transgene concentration, flanking matrix attachment regions, and RecA-coating on the efficiency of mouse transgenesis mediated by intracytoplasmic sperm injection. Biol Reprod 76:336–343

    Article  PubMed  CAS  Google Scholar 

  50. Porter SD, Hu J, Gilks CB (1999) Distal upstream tyrosinase S/MAR-containing sequence has regulatory properties specific to subsets of melanocytes. Dev Genet 25:40–48

    Article  PubMed  CAS  Google Scholar 

  51. Whitelaw CB, Grolli S, Accornero P, Donofrio G, Farini E, Webster J (2000) Matrix attachment region regulates basal beta-lactoglobulin transgene expression. Gene 244:73–80

    Article  PubMed  CAS  Google Scholar 

  52. Bessa J, Tena JJ, de la Calle-Mustienes E, Fernández-Miñán A, Naranjo S, Fernández A, Montoliu L, Akalin A, Lenhard B, Casares F, Gómez-Skarmeta JL (2009) Zebrafish enhancer detection (ZED) vector: a new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish. Dev Dyn 238:2409–2417

    Article  PubMed  CAS  Google Scholar 

  53. Moltó E, Fernández A, Montoliu L (2009) Boundaries in vertebrate genomes: different solutions to adequately insulate gene expression domains. Brief Funct Genomic Proteomic 8:283–296

    Article  PubMed  Google Scholar 

  54. Mountford P, Zevnik B, Düwel A, Nichols J, Li M, Dani C, Robertson M, Chambers I, Smith A (1994) Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc Natl Acad Sci USA 91:4303–4307

    Article  PubMed  CAS  Google Scholar 

  55. Gorski JA, Jones KR (1999) Efficient bicistronic expression of cre in mammalian cells. Nucleic Acids Res 27:2059–2061

    Article  PubMed  CAS  Google Scholar 

  56. Shaw-Jackson C, Michiels T (1999) Absence of internal ribosome entry site-mediated tissue specificity in the translation of a bicistronic transgene. J Virol 73:2729–2738

    PubMed  CAS  Google Scholar 

  57. Attal J, Théron MC, Rival S, Puissant C, Houdebine LM (2000) The efficiency of different IRESs (internal ribosomes entry site) in monocistronic mRNAS. Mol Biol Rep 27:21–26

    Article  PubMed  CAS  Google Scholar 

  58. Attal J, Theron MC, Puissant C, Houdebine LM (1999) Effect of intercistronic length on internal ribosome entry site (IRES) efficiency in bicistronic mRNA. Gene Expr 8:299–309

    PubMed  CAS  Google Scholar 

  59. Attal J, Théron MC, Houdebine LM (1999) The optimal use of IRES (internal ribosome entry site) in expression vectors. Genet Anal 15:161–165

    Article  PubMed  CAS  Google Scholar 

  60. Udvardy A, Maine E, Schedl P (1985) The 87A7 chromomere. dentification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains. J Mol Biol 185:341–358

    Article  PubMed  CAS  Google Scholar 

  61. Chung JH, Whiteley M, Felsenfeld G (1993) A 5′ element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila. Cell 74:505–514

    Article  PubMed  CAS  Google Scholar 

  62. Bell AC, Felsenfeld G (1999) Stopped at the border: boundaries and insulators. Curr Opin Genet Dev 9:191–198

    Article  PubMed  CAS  Google Scholar 

  63. Bell AC, West AG, Felsenfeld G (2001) Insulators and boundaries: versatile regulatory elements in the eukaryotic genome. Science 291:447–450

    Article  PubMed  CAS  Google Scholar 

  64. Lunyak VV, Prefontaine GG, Núñez E, Cramer T, Ju BG, Ohgi KA, Hutt K, Roy R, García-Díaz A, Zhu X, Yung Y, Montoliu L, Glass CK, Rosenfeld MG (2007) Developmentally regulated activation of a SINE B2 repeat as a domain boundary in organogenesis. Science 317:248–251

    Article  PubMed  CAS  Google Scholar 

  65. Furlan-Magaril M, Rebollar E, Guerrero G, Fernández A, Moltó E, González-Buendía E, Cantero M, Montoliu L, Recillas-Targa F (2011) An insulator embedded in the chicken {alpha}-globin locus regulates chromatin domain configuration and differential gene expression. Nucleic Acids Res 39:89–103

    Article  PubMed  CAS  Google Scholar 

  66. Taboit-Dameron F, Malassagne B, Viglietta C, Puissant C, Leroux-Coyau M, Chéreau C, Attal J, Weill B, Houdebine LM (1999) Association of the 5′HS4 sequence of the chicken beta-globin locus control region with human EF1 alpha gene promoter induces ubiquitous and high expression of human CD55 and CD59 cDNAs in transgenic rabbits. Transgenic Res 8:223–235

    Article  PubMed  CAS  Google Scholar 

  67. Potts W, Tucker D, Wood H, Martin C (2000) Chicken beta-globin 5′HS4 insulators function to reduce variability in transgenic founder mice. Biochem Biophys Res Commun 273:1015–1018

    Article  PubMed  CAS  Google Scholar 

  68. Rival-Gervier S, Pantano T, Viglietta C, Maeder C, Prince S, Attal J, Jolivet G, Houdebine LM (2003) The insulator effect of the 5′HS4 region from the beta-globin chicken locus on the rabbit WAP gene promoter activity in transgenic mice. Transgenic Res 12:723–730

    Article  PubMed  CAS  Google Scholar 

  69. Giraldo P, Rival-Gervier S, Houdebine LM, Montoliu L (2003) The potential benefits of insulators on heterologous constructs in transgenic animals. Transgenic Res 12:751–755

    Article  PubMed  CAS  Google Scholar 

  70. Recillas-Targa F, Valadez-Graham V, Farrell CM (2004) Prospects and implications of using chromatin insulators in gene therapy and transgenesis. Bioessays 26:796–807

    Article  PubMed  CAS  Google Scholar 

  71. Román AC, González-Rico FJ, Moltó E, Hernando H, Neto A, Vicente C, Ballestar E, Gómez-Skarmeta JL, Vavrova J, White RJ, Montoliu L, Fernández-Salguero PM (2011) Dioxin receptor and SLUG transcription factors regulate the insulator activity of B1 SINE retrotransposons via an RNA polymerase switch. Genome Res 21(3):422–432

    Article  PubMed  Google Scholar 

  72. Thorey IS, Ceceña G, Reynolds W, Oshima RG (1993) Alu sequence involvement in transcriptional insulation of the keratin 18 gene in transgenic mice. Mol Cell Biol 13:6742–6751

    PubMed  CAS  Google Scholar 

  73. Willoughby DA, Vilalta A, Oshima RG (2000) An Alu element from the K18 gene confers position-independent expression in transgenic mice. J Biol Chem 275:759–768

    Article  PubMed  CAS  Google Scholar 

  74. Montoliu L (2002) Gene transfer strategies in animal transgenesis. Cloning Stem Cells 4:39–46

    Article  PubMed  CAS  Google Scholar 

  75. Recillas-Targa F (2006) Multiple strategies for gene transfer, expression, knockdown, and chromatin influence in mammalian cell lines and transgenic animals. Mol Biotechnol 34:337–354

    Article  PubMed  CAS  Google Scholar 

  76. Jakobovits A, Moore AL, Green LL, Vergara GJ, Maynard-Currie CE, Austin HA et al (1993) Germ-line transmission and expression of a human-derived yeast artificial chromosome. Nature 362:255–258

    Article  PubMed  CAS  Google Scholar 

  77. Strauss WM, Dausman J, Beard C, Johnson C, Lawrence JB, Jaenisch R (1993) Germ line transmission of a yeast artificial chromosome spanning the murine alpha 1(I) collagen locus. Science 259:1904–1907

    Article  PubMed  CAS  Google Scholar 

  78. Lamb BT, Sisodia SS, Lawler AM, Slunt HH, Kitt CA, Kearns WG, Pearson PL, Price DL, Gearhart JD (1993) Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice. Nat Genet 5:22–30

    Article  PubMed  CAS  Google Scholar 

  79. Pearson BE, Choi TK (1993) Expression of the human beta-amyloid precursor protein gene from a yeast artificial chromosome in transgenic mice. Proc Natl Acad Sci USA 90:10578–10582

    Article  PubMed  CAS  Google Scholar 

  80. Giraldo P, Montoliu L (2002) Artificial chromosome transgenesis in pigmentary research. Pigment Cell Res 15:258–264

    Article  PubMed  CAS  Google Scholar 

  81. Schedl A, Beermann F, Thies E, Montoliu L, Kelsey G, Schütz G (1992) Transgenic mice generated by pronuclear injection of a yeast artificial chromosome. Nucleic Acids Res 20:3073–3077

    Article  PubMed  CAS  Google Scholar 

  82. Moreira PN, Pozueta J, Giraldo P, Gutiérrez-Adán A, Montoliu L (2006) Generation of yeast artificial chromosome transgenic mice by intracytoplasmic sperm injection. Methods Mol Biol 349:151–161

    PubMed  Google Scholar 

  83. Moreira PN, Pozueta J, Pérez-Crespo M, Valdivieso F, Gutiérrez-Adán A, Montoliu L (2007) Improving the generation of genomic-type transgenic mice by ICSI. Transgenic Res 16:163–168

    Article  PubMed  CAS  Google Scholar 

  84. Yang XW, Model P, Heintz N (1997) Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol 15:859–865

    Article  PubMed  CAS  Google Scholar 

  85. Camper SA, Saunders TL (2000) Transgenic rescue of mutant phenotypes using large DNA fragments. In: Accili D (ed) Genetic manipulation of receptor expression and function. Wiley, New York, NY, pp 1–22

    Google Scholar 

  86. Heintz N (2000) Analysis of mammalian central nervous system gene expression and function using bacterial artificial chromosome-mediated transgenesis. Hum Mol Genet 9:937–943

    Article  PubMed  CAS  Google Scholar 

  87. Van Keuren ML, Gavrilina GB, Filipiak WE, Zeidler MG, Saunders TL (2009) Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes. Transgenic Res 18:769–785

    Article  PubMed  CAS  Google Scholar 

  88. Giraldo P, Giménez E, Montoliu L (1999) The use of yeast artificial chromosomes in transgenic animals: expression studies of the tyrosinase gene in transgenic mice. Genet Anal 15:175–178

    Article  PubMed  CAS  Google Scholar 

  89. Testa G, Vintersten K, Zhang Y, Benes V, Muyrers JP, Stewart AF (2004) BAC engineering for the generation of ES cell-targeting constructs and mouse transgenes. Methods Mol Biol 256:123–139

    PubMed  CAS  Google Scholar 

  90. Ohtsuka M, Kimura M, Tanaka M, Inoko H (2009) Recombinant DNA technologies for construction of precisely designed transgene constructs. Curr Pharm Biotechnol 10:244–251

    Article  PubMed  CAS  Google Scholar 

  91. Heintz N (2001) BAC to the future: the use of bac transgenic mice for neuroscience research. Nat Rev Neurosci 2:861–870

    Article  PubMed  CAS  Google Scholar 

  92. Peterson KR (2003) Transgenic mice carrying yeast artificial chromosomes. Expert Rev Mol Med 5:1–25

    Article  PubMed  Google Scholar 

  93. Yang XW, Gong S (2005) An overview on the generation of BAC transgenic mice for neuroscience research. Curr Protoc Neurosci. Chapter 5:Unit 5.20

    Google Scholar 

  94. Deal KK, Cantrell VA, Chandler RL, Saunders TL, Mortlock DP, Southard-Smith EM (2006) Distant regulatory elements in a Sox10-beta GEO BAC transgene are required for expression of Sox10 in the enteric nervous system and other neural crest-derived tissues. Dev Dyn 235:1413–1432

    Article  PubMed  CAS  Google Scholar 

  95. Sparwasser T, Eberl G (2007) BAC to immunology–bacterial artificial chromosome-mediated transgenesis for targeting of immune cells. Immunology 121:308–313

    Article  PubMed  CAS  Google Scholar 

  96. Regales L, Giraldo P, García-Díaz A, Lavado A, Montoliu L (2003) Identification and functional validation of a 5′ upstream regulatory sequence in the human tyrosinase gene homologous to the locus control region of the mouse tyrosinase gene. Pigment Cell Res 16:685–692

    Article  PubMed  CAS  Google Scholar 

  97. Millot B, Montoliu L, Fontaine ML, Mata T, Devinoy E (2003) Hormone-induced modifications of the chromatin structure surrounding upstream regulatory regions conserved between the mouse and rabbit whey acidic protein genes. Biochem J 372(Pt 1):41–52

    Article  PubMed  CAS  Google Scholar 

  98. Montoliu L, Roy R, Regales L, García-Díaz A (2009) Design of vectors for transgene expression: the use of genomic comparative approaches. Comp Immunol Microbiol Infect Dis 32:81–90

    Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by grants to L.M. from the Spanish Ministry of Science and Innovation (MICINN; BIO2009-1297). E.M. is supported by CIBERER (ISCIII) and C.V-G. by the CSIC JAE Predoc program.

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, Darwin 3, 28049, Madrid, Spain

    Eduardo Moltó & Cristina Vicente-García

  2. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain

    Eduardo Moltó, Cristina Vicente-García & Lluis Montoliu

  3. Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, C/Darwin 3, 28049, Madrid, Spain

    Lluis Montoliu

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  1. Eduardo Moltó
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  2. Cristina Vicente-García
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  3. Lluis Montoliu
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Correspondence to Lluis Montoliu .

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  1. Div. Biology, California Institute of Technology, Pasadena, 91125, California, USA

    Shirley Pease

  2. Transgenic Animal Model Core, University of Michigan, Ann Arbor, 48109-5674, Michigan, USA

    Thomas L. Saunders

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Moltó, E., Vicente-García, C., Montoliu, L. (2011). Designing Transgenes for Optimal Expression. In: Pease, S., Saunders, T. (eds) Advanced Protocols for Animal Transgenesis. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-20792-1_3

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