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Generation of Transgenic Animals by Use of YACs

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Advanced Protocols for Animal Transgenesis

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

The use of genomic-type DNA constructs ensures optimal transgene expression, once inserted into the host genome, because their large size includes most if not all the regulatory elements that are needed for correct gene expression. Large heterologous DNA molecules can be easily manipulated in bacterial or yeast cells, through the use of bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs), respectively. YACs are the vectors that allow the manipulation of larger DNA molecules, in excess of 1 Mb (1,000 kb). Some mammalian loci (i.e., the APP locus, ~450 kb) greatly exceed the maximum size for inserts that can be accommodated by BACs. Therefore YACs are currently the only available robust and reliable solution for working with these large genes. In this chapter, we will describe several procedures directed towards the preparation of YAC DNA of a quality suitable for microinjection into mouse embryos, for the successful generation of transgenic mice.

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Abbreviations

AHM:

Acid hydrolyzed casein

BAC:

Bacterial artificial chromosome

ddH2O:

Double distilled water

DNA:

Deoxyribonucleic acid

EDTA:

Ethylenediaminetetraacetic acid

ES:

Embryonic stem cell

ICSI:

Intracytoplasmic sperm injection

LMP:

Low-melting point

Mb:

Megabase (= 1,000 kilobases)

PCR:

Polymerase chain reaction

PFGE:

Pulsed-field gel electrophoresis

RFLP:

Restriction fragment length polymorphism

SDS:

Sodium dodecyl sulfate

SNP:

Single nucleotide polymorphism

TAE:

Buffer solution containing a mixture of Tris base, acetic acid and EDTA

TBE:

Buffer solution containing a mixture of Tris base, boric acid and EDTA

YAC:

Yeast artificial chromosome

References

  1. 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 

  2. 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 

  3. Den Dunnen JT, Grootscholten PM, Dauwerse JG, Walker AP, Monaco AP, Butler R, Anand R, Coffey AJ, Bentley DR, Steensma HY et al (1992) Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. Hum Mol Genet 1(1):19–28

    Article  Google Scholar 

  4. Schedl A, Larin Z, Montoliu L, Thies E, Kelsey G, Lehrach H, Schütz G (1993) A method for the generation of YAC transgenic mice by pronuclear microinjection. Nucleic Acids Res 21:4783–4787

    Article  PubMed  CAS  Google Scholar 

  5. 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 

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

    Article  PubMed  Google Scholar 

  7. Forget BG (1993) YAC transgenes: bigger is probably better. Proc Natl Acad Sci USA 90:7909–7911

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  9. Fujiwara Y, Miwa M, Takahashi R, Hirabayashi M, Suzuki T, Ueda M (1997) Position-independent and high-level expression of human alpha-lactalbumin in the milk of transgenic rats carrying a 210-kb YAC DNA. Mol Reprod Dev 47:157–163

    Article  PubMed  CAS  Google Scholar 

  10. Takahashi R, Ito K, Fujiwara Y, Kodaira K, Kodaira K, Hirabayashi M, Ueda M (2000) Generation of transgenic rats with YACs and BACs: preparation procedures and integrity of microinjected DNA. Exp Anim 49:229–233

    Article  PubMed  CAS  Google Scholar 

  11. Takahashi R, Ueda M (2010) Generation of transgenic rats using YAC and BAC DNA constructs. Methods Mol Biol 597:93–108

    Article  PubMed  CAS  Google Scholar 

  12. Brem G, Besenfelder U, Aigner B, Müller M, Liebl I, Schütz G, Montoliu L (1996) YAC transgenesis in farm animals: rescue of albinism in rabbits. Mol Reprod Dev 44:56–62

    Article  PubMed  CAS  Google Scholar 

  13. Langford GA, Cozzi E, Yannoutsos N, Lancaster R, Elsome K, Chen P, White DJ (1996) Production of pigs transgenic for human regulators of complement activation using YAC technology. Transplant Proc 28:862–863

    PubMed  CAS  Google Scholar 

  14. Zhang XF, Wu GX, Chen JQ, Zhang AM, Liu SG, Jiao BH, Cheng GX (2005) Transfer of an expression YAC into goat fetal fibroblasts by cell fusion for mammary gland bioreactor. Biochem Biophys Res Commun 333:58–63

    Article  PubMed  CAS  Google Scholar 

  15. 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 

  16. 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 

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

    Article  PubMed  CAS  Google Scholar 

  18. Zou X, Xian J, Davies NP, Popov AV, Brüggemann M (1996) Dominant expression of a 1.3 Mb human Ig kappa locus replacing mouse light chain production. FASEB J 10:1227–1232

    PubMed  CAS  Google Scholar 

  19. Popov AV, Zou X, Xian J, Nicholson IC, Brüggemann M (1999) A human immunoglobulin lambda locus is similarly well expressed in mice and humans. J Exp Med 189:1611–1620

    Article  PubMed  CAS  Google Scholar 

  20. Davies NP, Huxley C (1996) YAC transfer into mammalian cells by cell fusion. Methods Mol Biol 54:281–292

    PubMed  CAS  Google Scholar 

  21. Strauss WM, Jaenisch R (1992) Molecular complementation of a collagen mutation in mammalian cells using yeast artificial chromosomes. EMBO J 11:417–422

    PubMed  CAS  Google Scholar 

  22. 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 

  23. Strauss WM (1996) Transfection of mammalian cells via lipofection. Methods Mol Biol 54:307–327

    PubMed  CAS  Google Scholar 

  24. 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 

  25. Montoliu L, Bock CT, Schütz G, Zentgraf H (1995) Visualization of large DNA molecules by electron microscopy with polyamines: application to the analysis of yeast endogenous and artificial chromosomes. J Mol Biol 246:486–492

    Article  PubMed  CAS  Google Scholar 

  26. 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 

  27. Moreira PN, Giraldo P, Cozar P, Pozueta J, Jiménez A, Montoliu L, Gutiérrez-Adán A (2004) Efficient generation of transgenic mice with intact yeast artificial chromosomes by intracytoplasmic sperm injection. Biol Reprod 71:1943–1947

    Article  PubMed  CAS  Google Scholar 

  28. 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 

  29. 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 

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

    Article  PubMed  CAS  Google Scholar 

  31. 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 

  32. Huxley C, Hagino Y, Schlessinger D, Olson MV (1991) The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion. Genomics 9:742–750

    Article  PubMed  CAS  Google Scholar 

  33. Schedl A, Grimes B, Montoliu L (1996) YAC transfer by microinjection. Methods Mol Biol 54:293–306

    PubMed  CAS  Google Scholar 

  34. Umland T, Montoliu L, Schütz G (1997) The use of yeast artificial chromosome for transgenesis. In: Houdebine LM (ed) Transgenic animals. Generation and use. Harwood Academic Publishers, Amsterdam, pp 289–298

    Google Scholar 

  35. Hiemisch H, Umland T, Montoliu L, Schütz G (1998) The generation of transgenic mice with yeast artificial chromosomes. In: Cid-Arregui A, Garcia-Carranca A (eds) Microinjection and transgenesis, strategies and protocols. Springer, Berlin, pp 297–308

    Chapter  Google Scholar 

  36. Shen S, Harmar A, Hastie N (2006) Modification and amplification of yeast artificial chromosomes. Methods Mol Biol 349:67–74

    PubMed  CAS  Google Scholar 

  37. 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 

  38. MacKenzie A (2006) Production of yeast artificial chromosome transgenic mice by pronuclear injection of one-cell embryos. Methods Mol Biol 349:139–150

    PubMed  CAS  Google Scholar 

  39. Peterson KR (2007) Preparation of intact yeast artificial chromosome DNA for transgenesis of mice. Nat Protoc 2:3009–3015

    Article  PubMed  CAS  Google Scholar 

  40. Jakobovits A, Lamb BT, Peterson KR (2000) Production of transgenic mice with yeast artificial chromosomes. Methods Mol Biol 136:435–453

    Google Scholar 

  41. Hamer L, Johnston M, Green ED (1995) Isolation of yeast artificial chromosomes free of endogenous yeast chromosomes: construction of alternate hosts with defined karyotypic alterations. Proc Natl Acad Sci USA 92:11706–11710

    Google Scholar 

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Author information

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

    Almudena Fernández & Diego Muñoz

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

    Almudena Fernández, Diego Muñoz & 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

Authors
  1. Almudena Fernández
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  2. Diego Muñoz
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  3. Lluis Montoliu
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Corresponding author

Correspondence to Lluis Montoliu .

Editor information

Editors and Affiliations

  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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© 2011 Springer-Verlag Berlin Heidelberg

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Fernández, A., Muñoz, D., Montoliu, L. (2011). Generation of Transgenic Animals by Use of YACs. 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_8

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  • DOI: https://doi.org/10.1007/978-3-642-20792-1_8

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-642-20791-4

  • Online ISBN: 978-3-642-20792-1

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