Transgene Recombineering in Bacterial Artificial Chromosomes

  • Michael G. Zeidler
  • Thomas L. SaundersEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1874)


Bacterial Artificial Chromosome (BAC) libraries are a valuable research resource. Any one of the clones in these libraries can carry hundreds of thousands of base pairs of genetic information. Often the entire coding sequence and significant upstream and downstream regions, including regulatory elements, can be found in a single BAC clone. BACs can be put to many uses, such as to study the function of human genes in knockout mice, to drive reporter gene expression in transgenic animals, and for gene discovery. In order to use BACs for experimental purposes it is often desirable to genetically modify them by introducing reporter elements or heterologous cDNA sequences. It is not feasible to use conventional DNA cloning approaches to modify BACs due to their size and complexity, thus a specialized field “recombineering” has developed to modify BAC clones through the use of homologous recombination in bacteria with short homology regions. Genetically engineered BACs can then be used in cell culture, mouse, or rat models to study cancer, neurology, and genetics.

Key words

BAC clone BAC transgenic BAC transgenesis Recombineering Transgenic mice 


  1. 1.
    Giraldo P, Montoliu L (2001) Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res 10:83–103CrossRefGoogle Scholar
  2. 2.
    Heaney JD, Bronson SK (2006) Artificial chromosome-based transgenes in the study of genome function. Mamm Genome 17:791–807CrossRefGoogle Scholar
  3. 3.
    Osoegawa K, Mammoser AG, Wu C, Frengen E, Zeng C, Catanese JJ, de Jong PJ (2001) A bacterial artificial chromosome library for sequencing the complete human genome. Genome Res 11:483–496CrossRefGoogle Scholar
  4. 4.
    Osoegawa K, Tateno M, Woon PY, Frengen E, Mammoser AG, Catanese JJ, Hayashizaki Y, de Jong PJ (2000) Bacterial artificial chromosome libraries for mouse sequencing and functional analysis. Genome Res 10:116–128PubMedPubMedCentralGoogle Scholar
  5. 5.
    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 18:769–785CrossRefGoogle Scholar
  6. 6.
    Alzhanov D, Rotwein P (2016) Characterizing a distal muscle enhancer in the mouse Igf2 locus. Physiol Genomics 48:167–172CrossRefGoogle Scholar
  7. 7.
    Deal KK, Cantrell VA, Chandler RL, Saunders TL, Mortlock DP, Southard-Smith EM (2006) Distant regulatory elements in a Sox10-betaGEO BAC transgene are required for expression of Sox10 in the enteric nervous system and other neural crest-derived tissues. Dev Dyn 235:1413–1432CrossRefGoogle Scholar
  8. 8.
    Dunnick WA, Shi J, Fontaine C, Collins JT (2013) Transgenes of the mouse immunoglobulin heavy chain locus, lacking distal elements in the 3′ regulatory region, are impaired for class switch recombination. PLoS One 8:e55842CrossRefGoogle Scholar
  9. 9.
    Davis SW, Keisler JL, Pérez-Millán MI, Schade V, Camper SA (2016) All hormone-producing cell types of the pituitary intermediate and anterior lobes derive from Prop1 expressing progenitors. Endocrinology 157:1385–1396CrossRefGoogle Scholar
  10. 10.
    Jones JM, Datta P, Srinivasula SM, Ji W, Gupta S, Zhang Z, Davies E, Hajnoczky G, Saunders TL, Van Keuren ML, Fernandes-Alnemri T, Meisler MH, Alnemri ES (2003) Loss of Omi mitochondrial protease activity causes the neuromuscular disorder of mnd2 mutant mice. Nature 425:721–727CrossRefGoogle Scholar
  11. 11.
    Khoriaty R, Everett L, Chase J, Zhu G, Hoenerhoff M, McKnight B, Vasievich MP, Zhang B, Tomberg K, Williams J, Maillard I, Ginsburg D (2016) Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice. Sci Rep 6:27802CrossRefGoogle Scholar
  12. 12.
    Probst FJ, Fridell RA, Raphael Y, Wang A, Liang Y, Morell RJ, Touchman JW, Lyons RH, Noben-Trauth K, Friedman TB, Camper SA (1998) Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene. Science 280:1444–1447CrossRefGoogle Scholar
  13. 13.
    Antoch MP, Song EJ, Chang AM, Vitaterna MH, Zhao Y, Wilsbacher LD, Sangoram AM, King DP, Pinto LH, Takahashi JS (1997) Functional identification of the mouse circadian clock gene by transgenic BAC rescue. Cell 89:655–667CrossRefGoogle Scholar
  14. 14.
    Hu Y, Smith DE (2016) Species differences in the pharmacokinetics of cefadroxil as determined in wildtype and humanized PepT1 mice. Biochem Pharmacol 107:81–90CrossRefGoogle Scholar
  15. 15.
    Mensah-Osman E, Labut E, Zavros Y, El-Zaatari M, Law DJ, Merchant JL (2008) Regulated expression of the human gastrin gene in mice. Regul Pept 151:115–122CrossRefGoogle Scholar
  16. 16.
    Sarsero JP, Holloway TP, Li L, Finkelstein DI, Ioannou PA (2014) Rescue of the Friedreich ataxia knockout mutation in transgenic mice containing an FXN-EGFP genomic reporter. PLoS One 9:e93307CrossRefGoogle Scholar
  17. 17.
    Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N (2003) A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917–925CrossRefGoogle Scholar
  18. 18.
    Valjent E, Bertran-Gonzalez J, Hervé D, Fisone G, Girault JA (2009) Looking BAC at striatal signaling: cell-specific analysis in new transgenic mice. Trends Neurosci 32:538–547CrossRefGoogle Scholar
  19. 19.
    Witten IB, Steinberg EE, Lee SY, Davidson TJ, Zalocusky KA, Brodsky M, Yizhar O, Cho SL, Gong S, Ramakrishnan C, Stuber GD, Tye KM, Janak PH, Deisseroth K (2011) Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement. Neuron 72:721–733CrossRefGoogle Scholar
  20. 20.
    Keegan CE, Karolyi IJ, Knapp LT, Bourbonais FJ, Camper SA, Seasholtz AF (1994) Expression of corticotropin-releasing hormone transgenes in neurons of adult and developing mice. Mol Cell Neurosci 5:505–514CrossRefGoogle Scholar
  21. 21.
    Alon T, Zhou L, Pérez CA, Garfield AS, Friedman JM, Heisler LK (2009) Transgenic mice expressing green fluorescent protein under the control of the corticotropin-releasing hormone promoter. Endocrinology 150:5626–5632CrossRefGoogle Scholar
  22. 22.
    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 235:1413–2432CrossRefGoogle Scholar
  23. 23.
    Dunnick WA, Shi J, Holden V, Fontaine C, Collins JT (2011) The role of germline promoters and I exons in cytokine-induced gene-specific class switch recombination. J Immunol 186:350–358CrossRefGoogle Scholar
  24. 24.
    Lehoczky JA, Innis JW (2008) BAC transgenic analysis reveals enhancers sufficient for Hoxa13 and neighborhood gene expression in mouse embryonic distal limbs and genital bud. Evol Dev 10:421–432CrossRefGoogle Scholar
  25. 25.
    Pérez-Millán MI, Zeidler MG, Saunders TL, Camper SA, Davis SW (2013) Efficient, specific, developmentally appropriate cre-mediated recombination in anterior pituitary gonadotropes and thyrotropes. Genesis 51:785–792CrossRefGoogle Scholar
  26. 26.
    Yu S, Zhou X, Hsiao JJ, Yu D, Saunders TL, Xue HH (2012) Fidelity of a BAC-EGFP transgene in reporting dynamic expression of IL-7Rα in T cells. Transgenic Res 21:201–215CrossRefGoogle Scholar
  27. 27.
    Lehoczky JA, Thomas PE, Patrie KM, Owens KM, Villarreal LM, Galbraith K, Washburn J, Johnson CN, Gavino B, Borowsky AD, Millen KJ, Wakenight P, Law W, Van Keuren ML, Gavrilina G, Hughes ED, Saunders TL, Brihn L, Nadeau JH, Innis JW (2013) A novel intergenic ETnII-β insertion mutation causes multiple malformations in polypodia mice. PLoS Genet 9:e1003967CrossRefGoogle Scholar
  28. 28.
    Watson CL, Mahe MM, Múnera J, Howell JC, Sundaram N, Poling HM, Schweitzer JI, Vallance JE, Mayhew CN, Sun Y, Grabowski G, Finkbeiner SR, Spence JR, Shroyer NF, Wells JM, Helmrath MA (2014) An in vivo model of human small intestine using pluripotent stem cells. Nat Med 20:1310–1314CrossRefGoogle Scholar
  29. 29.
    Albertsen HM, Abderrahim H, Cann HM, Dausset J, Le Paslier D, Cohen D (1990) Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents. Proc Natl Acad Sci U S A 87:4256–4260CrossRefGoogle Scholar
  30. 30.
    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 16:163–168CrossRefGoogle Scholar
  31. 31.
    Brandt W, Khandekar M, Suzuki N, Yamamoto M, Lim KC, Engel JD (2008) Defining the functional boundaries of the Gata2 locus by rescue with a linked bacterial artificial chromosome transgene. J Biol Chem 283:8976–8983CrossRefGoogle Scholar
  32. 32.
    Sopher BL, La Spada AR (2006) Efficient recombination-based methods for bacterial artificial chromosome fusion and mutagenesis. Gene 12(371):136–143CrossRefGoogle Scholar
  33. 33.
    Montoliu L, Bock CT, Schutz 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 246:486–492CrossRefGoogle Scholar
  34. 34.
    Montigny WJ, Phelps SF, Illenye S, Heintz NH (2003) Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells. BioTechniques 35:796–807CrossRefGoogle Scholar
  35. 35.
    Copeland NG, Jenkins NA, Court DL (2001) Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet 2:769–779CrossRefGoogle Scholar
  36. 36.
    Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361–388CrossRefGoogle Scholar
  37. 37.
    Zhang Y, Muyrers JP, Testa G, Stewart AF (2000) DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol 18:1314–1317CrossRefGoogle Scholar
  38. 38.
    Auwerx J, Avner P, Baldock R, Ballabio A, Balling R, Barbacid M, Berns A, Bradley A, Brown S, Carmeliet P, Chambon P, Cox R, Davidson D, Davies K, Duboule D, Forejt J, Granucci F, Hastie N, de Angelis MH, Jackson I, Kioussis D, Kollias G, Lathrop M, Lendahl U, Malumbres M, von Melchner H, Müller W, Partanen J, Ricciardi-Castagnoli P, Rigby P, Rosen B, Rosenthal N, Skarnes B, Stewart AF, Thornton J, Tocchini-Valentini G, Wagner E, Wahli W, Wurst W (2004) The European dimension for the mouse genome mutagenesis program. Nat Genet 36:925–927CrossRefGoogle Scholar
  39. 39.
    International Mouse Knockout Consortium, Collins FS, Rossant J, Wurst W (2007) A mouse for all reasons. Cell 128:9–13CrossRefGoogle Scholar
  40. 40.
    Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W, Iyer V, Mujica AO, Thomas M, Harrow J, Cox T, Jackson D, Severin J, Biggs P, Fu J, Nefedov M, de Jong PJ, Stewart AF, Bradley A (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474:337–342CrossRefGoogle Scholar
  41. 41.
    Testa G, Zhang Y, Vintersten K, Benes V, Pijnappel WW, Chambers I, Smith AJ, Smith AG, Stewart AF (2003) Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat Biotechnol 21:443–447CrossRefGoogle Scholar
  42. 42.
    Valenzuela DM, Murphy AJ, Frendewey D, Gale NW, Economides AN, Auerbach W, Poueymirou WT, Adams NC, Rojas J, Yasenchak J, Chernomorsky R, Boucher M, Elsasser AL, Esau L, Zheng J, Griffiths JA, Wang X, Su H, Xue Y, Dominguez MG, Noguera I, Torres R, Macdonald LE, Stewart AF, DeChiara TM, Yancopoulos GD (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21:652–659CrossRefGoogle Scholar
  43. 43.
    Hu Y, Xie Y, Wang Y, Chen X, Smith DE (2014) Development and characterization of a novel mouse line humanized for the intestinal peptide transporter PEPT1. Mol Pharm 11:3737–3746CrossRefGoogle Scholar
  44. 44.
    Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG (2005) Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 33:e36CrossRefGoogle Scholar
  45. 45.
    Filipiak WE, Saunders TL (2006) Advances in transgenic rat production. Transgenic Res 15:673–686CrossRefGoogle Scholar
  46. 46.
    Becker K, Jerchow B (2011) Generation of transgenic mice by pronuclear microinjection. In: Pease S, Saunders TL (eds) Advanced protocols for animal Transgenesis: an ISTT manual. Springer-Verlag, BerlinGoogle Scholar
  47. 47.
    Ralser M, Querfurth R, Warnatz HJ, Lehrach H, Yaspo ML, Krobitsch S (2006) An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun 347:747–751CrossRefGoogle Scholar
  48. 48.
    St-Pierre F, Cui L, Priest DG, Endy D, Dodd IB, Shearwin KE (2013) One-step cloning and chromosomal integration of DNA. ACS Synth Biol 2:537–541CrossRefGoogle Scholar
  49. 49.
    Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134CrossRefGoogle Scholar
  50. 50.
    Stratman JL, Barnes WM, Simon TC (2003) Universal PCR genotyping assay that achieves single copy sensitivity with any primer pair. Transgenic Res 12:521–552CrossRefGoogle Scholar
  51. 51.
    Lee EC, Yu D, Martinez de Velasco J, Tessarollo L, Swing DA, Court DL, Jenkins NA, Copeland NG (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56–65CrossRefGoogle Scholar
  52. 52.
    Schnütgen F, Ghyselinck NB (2007) Adopting the good reFLEXes when generating conditional alterations in the mouse genome. Transgenic Res 16:405–413CrossRefGoogle Scholar
  53. 53.
    Chuang K, Nguyen E, Sergeev Y, Badea TC (2016) Novel heterotypic Rox sites for combinatorial dre recombination strategies. G3 (Bethesda) 6:559–571CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.University of Michigan Transgenic Animal Model CoreAnn ArborUSA
  2. 2.Division of Genetic Medicine, Department of Internal MedicineUniversity of MichiganAnn ArborUSA

Personalised recommendations