BAC transgenics (rat)
Bacterial artificial chromosomes or BACs have a payload capacity of several hundred kilobases and offer advantages over conventional transgenes because BAC transgenes contain additional sequence context that typically insulates the gene of interest from chromosomal positional effects. This large sequence around the transgene circuit helps to maintain accurate transcription so that physiological levels of tissue-specific transcriptional control are recapitulated, and the extensive BAC sequence buffers against premature transgene silencing often seen in conventional transgene arrays. BAC vectors can be modified or customized by recombineering protocols.
Recombineering is a highly efficient and precise method used for the generation of seamlessly mutated bacterial artificial chromosomes (BACs) including insertion of point mutations, protein tags, landing pads and reporter genes. Our BAC recombineering service offers counterselection approaches, inactivation of non-relevant genes on the BAC to prevent overexpression, gene replacement for humanization and BAC reduction by shaving. Our recombineering portiolo consists of successful design of complex genetic circuits, incorporation of site-specific recombinases such as Cre, CreERt2, flippase (Flp/Flpe/Flpo), Dre, VCre, Vika, Nigri & Panto, usage of heterotypic recombinase sites such as lox66/lox2272, Frt3/Frt5/Frt14/15, rox/rox12, recycling of antibiotic cassettes for selection with ampicillin, blasticidin, carbenicillin, chloramphenicol, gentamycin, hygromycin, nourseothricin (clonNAT), puromycin, spectinomycin, streptomycin, tetracycline, trimethoprim and zeocin/bleocin/phleomycin. We include up to 2 recombineering steps for our basic service with NovoHelix custom model generation (animal model). Basic service includes selection with ampicillin, carbenicillin, chloramphenicol, spectinomycin, tetracycline or trimethoprim. Additional charges may incur for more recombineering steps and selection with antibiotics not included in our basic service.
model generation
support services
technology
references
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model generation
Service
Catalog Nr
Service Description
Timeline
Deliverables
Pricing
Rat Sprague Dawley - BAC transgene pronuclear microinjection
RBAC001
Rat Sprague Dawley strain BAC transgene pronuclear microinjection
(includes BAC purification and founder genotyping)
3 - 4 months
2 BAC founders
Custom rat strain - BAC transgene pronuclear microinjection
RBAC002
Brown Norway, Fischer, Lewis, Long-Evans, Sprague Dawley, Wistar, Zucker
Other custom strains available upon request for BAC transgene pronuclear microinjection
(includes BAC purification and founder genotyping)
3 - 4 months
2 BAC founders
support services
Service
Catalog Nr
Service Description
Timeline
Deliverables
Pricing
BAC recombineering (in conjunction with NovoHelix animal model generation)
RBAC003
We include up to 2 recombineering steps for our basic service with NovoHelix custom model generation (animal model). Basic service includes selection with ampicillin, carbenicillin, chloramphenicol, spectinomycin, tetracycline or trimethoprim. Additional charges may incur for more recombineering steps and selection with antibiotics not included in our basic service. BAC purification service is included with this option.
2 - 8 weeks
E. coli strain containing modified BAC as glycerol stock or stab culture or 1-10 micrograms of modified supercoiled BAC
$3,150.00
BAC recombineering, basic
RBAC004
We include up to 2 recombineering steps for our basic service. Basic service includes selection with ampicillin, carbenicillin, chloramphenicol, kanamycin, spectinomycin, tetracycline or trimethoprim. Additional charges may incur for more recombineering steps and selection with antibiotics not included in our basic service. The modified BAC will be provided in the E. coli strain as a glycerol stock or stab culture.
2 - 4 weeks
E. coli strain containing modified BAC as glycerol stock or stab culture
$4,800.00
BAC recombineering - advanced
RBAC005
Advanced recombineering service includes steps that supercede 3 or more recombineering reactions and includes selection steps with non-standard antibiotics such as blasticidin, gentamycin, hygromycin, nourseothricin (clonNAT), puromycin, streptomycin and zeocin/bleocin/phleomycin or others. The advanced recombineering service route also includes seamless knockin of large cassettes > 3 kb and development of screening assays for E. coli specific-strain generation. NovoHelix has pioneered BAC shaving and deleted tens of kilobases from an existing BAC to remove extraneous sequences and genes linked on the same chromosome. The modified BAC will be provided in the E. coli strain as a glycerol stock or stab culture.
3 - 8 weeks
E. coli strain containing modified BAC as glycerol stock or stab culture
BAC purification & validation
RBAC006
Purification of supercoiled BACs by a proprietary anion exchange (AEX) chromatography, validation by PCR screening around the BAC at ~20-kb intervals and validation by restriction digestion and analysis by pulse-field gel electrophoresis (PFGE). Endotoxin levels are generally very low (0.05 – 0.5 ng LPS/µg) via our proprietary extraction method and are adequate for sensitive applications such as zygotic microinjection of mammalian embryos. The BAC is predominantly in its supercoiled topology and free of RNA and protein contamination. Linearization with a homing nuclease such as PI-SceI and drop dialysis in 1x TE or microinjection buffer is included in the purification service. Validation by PCR screening at higher resolution (5-kb, 10-kb) or additional restriction digestions (greater than 3) is available with commensurate fee schedule.
2-3 days
10-100 micrograms of supercoiled BAC
$1,850.00
Genotyping Assay Development - BACs
RBAC007
BACs are generally between 100-300 kb and their sheer size requires additional genotyping assays to determine if the full-length or partial BAC has integrated into the BAC transgenic founder. NovoHelix offers a genotyping service to help users develop robust genotyping protocols for screening animals after breeding and expansion of individual BAC founder lines.
genotyping protocol
$750.00
Rat colony husbandry & management
RBAC008
Rat colony management including breeding, weaning, sampling, monitoring, per cage per day for the production of founders and F-generation pups
technology
references
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Yang XW, Model P, Heintz N. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol. 1997 Sep;15(9):859-65. PubMed PMID: 9306400.
Zhang Y, Buchholz F, Muyrers JP, Stewart AF. A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet. 1998 Oct;20(2):123-8. PubMed PMID: 9771703.
Zhang Y, Muyrers JP, Testa G, Stewart AF. DNA cloning by homologous recombination in Escherichia coli. Nat Biotechnol. 2000 Dec;18(12):1314-7. PubMed PMID: 11101815.
Chrast R, Scott HS, Antonarakis SE. Linearization and purification of BAC DNA for the development of transgenic mice. Transgenic Res. 1999 Apr;8(2):147-50. PubMed PMID: 10481314.
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Bird AW, Erler A, Fu J, Hériché JK, Maresca M, Zhang Y, Hyman AA, Stewart AF. High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes. Nat Methods. 2011 Dec 4;9(1):103-9. doi: 10.1038/nmeth.1803. PubMed PMID: 22138824.
Chan W, Costantino N, Li R, Lee SC, Su Q, Melvin D, Court DL, Liu P. A recombineering based approach for high-throughput conditional knockout targeting vector construction. Nucleic Acids Res. 2007;35(8):e64. Epub 2007 Apr 10. PubMed PMID: 17426124; PubMed Central PMCID: PMC1885671.
Fu J, Bian X, Hu S, Wang H, Huang F, Seibert PM, Plaza A, Xia L, Müller R, Stewart AF, Zhang Y. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat Biotechnol. 2012 May;30(5):440-6. doi: 10.1038/nbt.2183. PubMed PMID: 22544021.
Thomason LC, Costantino N, Court DL. Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli. MBio. 2016 Sep 13;7(5). pii: e01443-16. doi:10.1128/mBio.01443-16. PubMed PMID: 27624131; PubMed Central PMCID: PMC5021808.
Egbert RG, Rishi HS, Adler BA, McCormick DM, Toro E, Gill RT, Arkin AP. A versatile platform strain for high-fidelity multiplex genome editing. Nucleic Acids Res. 2019 Apr 8;47(6):3244-3256. doi: 10.1093/nar/gkz085. PubMed PMID: 30788501; PubMed Central PMCID: PMC6451135.
Pines G, Freed EF, Winkler JD, Gill RT. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination. ACS Synth Biol. 2015 Nov 20;4(11):1176-85. doi: 10.1021/acssynbio.5b00009. Epub 2015 Apr 27. Review. PubMed PMID: 25856528.
Reynolds TS, Gill RT. Quantifying Impact of Chromosome Copy Number on Recombination in Escherichia coli. ACS Synth Biol. 2015 Jul 17;4(7):776-80. doi: 10.1021/sb500338g. Epub 2015 Mar 19. PubMed PMID: 25763604.
Wannier TM, Nyerges A, Kuchwara HM, Czikkely M, Balogh D, Filsinger GT, Borders NC, Gregg Cj, Lajoie MJ, Rios X, Pal C, Church GM. Improved bacterial recombineering by parallelized protein discovery. bioRxiv. 2020 Jan 16. doi: 10.1101/2020.01.14.906594.
Chuang K, Nguyen E, Sergeev Y, Badea TC. Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies. G3 (Bethesda). 2015 Dec 29;6(3):559-71. doi: 10.1534/g3.115.025841. PubMed PMID: 26715092; PubMed Central PMCID: PMC4777119.
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