Transgenic Research

, Volume 18, Issue 5, pp 769–785 | Cite as

Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

  • Margaret L. Van Keuren
  • Galina B. Gavrilina
  • Wanda E. Filipiak
  • Michael G. Zeidler
  • Thomas L. SaundersEmail author
Original Paper


Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.


Mice, Transgenic Gene transfer techniques Chromosomes, Artificial, Bacterial BAC Electrophoresis, Gel, Pulsed-field DNA Gene Expression 



We thank Tina Jones and Corey Ziebell for their management of the transgenic production mouse colonies and we thank Susan Allen for her editorial assistance. The Transgenic Animal Model Core of the University of Michigan’s Biomedical Research Core Facilities is supported by the University of Michigan Cancer Center (NIH CA046592), the University of Michigan Rheumatic Diseases Center Core (NIH AR048310), the University of Michigan Gastrointestinal Peptide Research Center (NIH DK034933), and the Nathan Shock Center for the Biology of Aging (NIH AG013283).


  1. 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 89:655–667. doi: 10.1016/S0092-8674(00)80246-9 PubMedCrossRefGoogle Scholar
  2. Baoan J, Song J, Tsou L, Bi Y, Gaiser S, Mortensen R, Logsdon C (2008) Robust acinar cell transgene expression of CreErT via BAC recombineering. Genesis 35:390–395Google Scholar
  3. Bishop JO (1996) Chromosomal insertion of foreign DNA. Reprod Nutr Dev 36:607–618PubMedGoogle Scholar
  4. Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD (1985) Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci USA 82:4438–4442. doi: 10.1073/pnas.82.13.4438 PubMedCrossRefGoogle Scholar
  5. Bronson SK, Plaehn EG, Kluckman KD, Hagaman JR, Maeda N, Smithies O (1996) Single-copy transgenic mice with chosen-site integration. Proc Natl Acad Sci USA 93:9067–9072. doi: 10.1073/pnas.93.17.9067 PubMedCrossRefGoogle Scholar
  6. Callow MJ, Stoltzfus LJ, Lawn RM, Rubin EM (1994) Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice. Proc Natl Acad Sci USA 91:2130–2134. doi: 10.1073/pnas.91.6.2130 PubMedCrossRefGoogle Scholar
  7. 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-Liss, New York, pp 1–22Google Scholar
  8. Chandler KJ, Chandler RL, Broeckelmann EM, Hou Y, Southard-Smith EM, Mortlock DP (2007) Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression. Mamm Genome 18:693–708. doi: 10.1007/s00335-007-9056-y PubMedCrossRefGoogle Scholar
  9. Chiesa G, Johnson DF, Yao Z, Innerarity TL, Mahley RW, Young SG, Hammer RH, Hobbs HH (1993) Expression of human apolipoprotein B100 in transgenic mice. Editing of human apolipoprotein B100 mRNA. J Biol Chem 268:23747–23750PubMedGoogle Scholar
  10. Cole KD, Tellez CM (2002) Separation of large circular DNA by electrophoresis in agarose gels. Biotechnol Prog 18:82–87. doi: 10.1021/bp010135o PubMedCrossRefGoogle Scholar
  11. Court DL, Sawitzke JA, Thomason LC (2002) Genetic engineering using homologous recombination. Annu Rev Genet 36:361–388. doi: 10.1146/annurev.genet.36.061102.093104 PubMedCrossRefGoogle Scholar
  12. 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 235:1413–1432. doi: 10.1002/dvdy.20769 PubMedCrossRefGoogle Scholar
  13. Dunnick WA, Shi J, Graves KA, Collins JT (2004) Germline transcription and switch recombination of a transgene containing the entire H chain constant region locus: effect of a mutation in a STAT6 binding site in the gamma1 promoter. J Immun 173:5531–5539PubMedGoogle Scholar
  14. Dunnick WA, Shi J, Graves KA, Collins JT (2005) The 3′ end of the heavy chain constant region locus enhances germline transcription and switch recombination of the four gamma genes. J Exp Med 201:1459–1466. doi: 10.1084/jem.20041988 PubMedCrossRefGoogle Scholar
  15. Fiorenza MT, Bevilacqua A, Bevilacqua S, Mangia F (2001) Growing dictyate oocytes, but not early preimplantation embryos, of the mouse display high levels of DNA homologous recombination by single-strand annealing and lack DNA nonhomologous end joining. Dev Biol 233:214–224. doi: 10.1006/dbio.2001.0199 PubMedCrossRefGoogle Scholar
  16. Giraldo P, Montoliu L (2001) Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res 10:83–103. doi: 10.1023/A:1008918913249 PubMedCrossRefGoogle Scholar
  17. Hammer RE, Krumlauf R, Camper SA, Brinster RL, Tilghman SM (1987) Diversity of alpha-fetoprotein gene expression in mice is generated by a combination of separate enhancer elements. Science 235:53–58. doi: 10.1126/science.2432657 PubMedCrossRefGoogle Scholar
  18. Jones JM, Datta P, Srinivasula SM, Ji W, Gupta S, Zhang Z, Davies E, Hajnocyzky SaundersTL, 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 425:721–727. doi: 10.1038/nature02052 PubMedCrossRefGoogle Scholar
  19. Keegan CE, Karolyi IJ, Burrows H, Camper SA, Seasholtz AF (1994) Homologous recombination in fertilized mouse eggs and assessment of heterologous LCR function. Transgenics 1:439–449Google Scholar
  20. Kjer-Nielsen L, Holmberg K, Perera JD, McCluskey J (1992) Impaired expression of chimaeric major histocompatibility complex transgenes associated with plasmid sequences. Transgenic Res 1:182–187. doi: 10.1007/BF02522537 PubMedCrossRefGoogle Scholar
  21. Krebs CJ, Larkins LK, Price R, Tullis KM, Miller RD, Robins DM (2003) Regulator of sex-limitation (Rsl) encodes a pair of KRAB zinc-finger genes that control sexually dimorphic liver gene expression. Genes Dev 17:2664–2674. doi: 10.1101/gad.1135703 PubMedCrossRefGoogle Scholar
  22. Larin Z, Monaco AP, Lehrach H (1991) Yeast artificial chromosome libraries containing large inserts from mouse and human DNA. Proc Natl Acad Sci USA 88:4123–4127. doi: 10.1073/pnas.88.10.4123 PubMedCrossRefGoogle Scholar
  23. 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 10:421–432. doi: 10.1111/j.1525-142X.2008.00253.x PubMedCrossRefGoogle Scholar
  24. Lo CW (1986) Localization of low abundance DNA sequences in tissue sections by in situ hybridization. J Cell Sci 81:143–162PubMedGoogle Scholar
  25. Luciw PA, Bishop JM, Varmus HE, Capecchi MR (1983) Location and function of retroviral and SV40 sequences that enhance biochemical transformation after microinjection of DNA. Cell 33:705–716PubMedCrossRefGoogle Scholar
  26. 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 151:115–122. doi: 10.1016/j.regpep.2008.03.009 PubMedCrossRefGoogle Scholar
  27. 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 246:486–492. doi: 10.1006/jmbi.1994.0100 PubMedCrossRefGoogle Scholar
  28. Nielsen LB, McCormick SP, Pierotti V, Tam C, Gunn MD, Shizuya H, Young SG (1997) Human apolipoprotein B transgenic mice generated with 207- and 145-kilobase pair bacterial artificial chromosomes. Evidence that a distant 5′-element confers appropriate transgene expression in the intestine. J Biol Chem 272:29752–29758. doi: 10.1074/jbc.272.47.29752 PubMedCrossRefGoogle Scholar
  29. Okita K, Ichisaka T, Yamanaka S (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448:313–317. doi: 10.1038/nature05934 PubMedCrossRefGoogle Scholar
  30. Oliver ER, Saunders TL, Tarlé SA, Glaser T (2004) Ribosomal protein L24 defect in Belly spot and tail (Bst), a mouse Minute. Development 131:3907–3920. doi: 10.1242/dev.01268 PubMedCrossRefGoogle Scholar
  31. 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 10:116–128PubMedGoogle Scholar
  32. Probst FJ, Fridell RA, Raphael Y, Saunders TL, 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 280:1444–1447. doi: 10.1126/science.280.5368.1444 PubMedCrossRefGoogle Scholar
  33. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491. doi: 10.1126/science.2448875 PubMedCrossRefGoogle Scholar
  34. Schedl A, Larin Z, Montoliu L, Thies E, Kelsey G, Lehrach H, Schütz G (1993a) A method for the generation of YAC transgenic mice by pronuclear microinjection. Nucleic Acids Res 21:4783–4787. doi: 10.1093/nar/21.20.4783 PubMedCrossRefGoogle Scholar
  35. Schedl A, Montoliu L, Kelsey G, Schutz G (1993b) A yeast artificial chromosome covering the tyrosinase region confers copy number dependent expression in transgenic mice. Nature 362:258–261. doi: 10.1038/362258a0 PubMedCrossRefGoogle Scholar
  36. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517. doi: 10.1016/S0022-2836(75)80083-0 PubMedCrossRefGoogle Scholar
  37. Sparwasser T, Gong S, Li JY, Eberl G (2004) General method for the modification of different BAC types and the rapid generation of BAC transgenic mice. Genesis 38:39–50. doi: 10.1002/gene.10249 PubMedCrossRefGoogle Scholar
  38. Sun H, Yang TL, Yang A, Wang X, Ginsburg D (2003) The murine platelet and plasma factor V pools are biosynthetically distinct and sufficient for minimal hemostasis. Blood 102:2856–2861. doi: 10.1182/blood-2003-04-1225 PubMedCrossRefGoogle Scholar
  39. Tacken PJ, van der Zee A, Beumer TL, Florijn RJ, Gijpels MJ, Havekes LM, Frants RR, van Dijk KW, Hofker MH (2001) Effective generation of very low density lipoprotein receptor transgenic mice by overlapping genomic DNA fragments: high testis expression and disturbed spermatogenesis. Transgenic Res 10:211–221. doi: 10.1023/A:1016682520887 PubMedCrossRefGoogle Scholar
  40. 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 21:443–447. doi: 10.1038/nbt804 PubMedCrossRefGoogle Scholar
  41. Townes TM, Lingrel JB, Chen HY, Brinster RL, Palmiter RD (1985) Erythroid-specific expression of human beta-globin genes in transgenic mice. EMBO J 4:1715–1723PubMedGoogle Scholar
  42. Wagner SD, Gross G, Cook GP, Davies SL, Neuberger MS (1996) Antibody expression from the core region of the human IgH locus reconstructed in transgenic mice using bacteriophage P1 clones. Genomics 35:405–414. doi: 10.1006/geno.1996.0379 PubMedCrossRefGoogle Scholar
  43. Wang M, Lai E (1995) Pulsed field separation of large supercoiled and open-circular DNAs and its application to bacterial artificial chromosome cloning. Electrophoresis 16:1–7. doi: 10.1002/elps.1150160102 PubMedCrossRefGoogle Scholar
  44. Wilson C, Bellen HJ, Gehring WJ (1990) Position effects on eukaryotic gene expression. Annu Rev Cell Biol 6:679–714. doi: 10.1146/annurev.cb.06.110190.003335 PubMedCrossRefGoogle Scholar
  45. Zar JH (1998) Biostatistical analysis, 4th edn. Prentice Hall, Upper Saddle RiverGoogle Scholar
  46. Zeller W, Meier G, Burki K, Panoussis B (1998) Adverse effects of tribromoethanol as used in the production of transgenic mice. Lab Anim 32:407–413. doi: 10.1258/002367798780599811 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Margaret L. Van Keuren
    • 1
  • Galina B. Gavrilina
    • 1
  • Wanda E. Filipiak
    • 1
  • Michael G. Zeidler
    • 1
  • Thomas L. Saunders
    • 1
    • 2
    Email author
  1. 1.Transgenic Animal Model CoreUniversity of Michigan Medical SchoolAnn ArborUSA
  2. 2.Department of Internal Medicine, Division of Molecular Medicine and GeneticsUniversity of Michigan Medical SchoolAnn ArborUSA

Personalised recommendations