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BAC transgenics - rat
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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. 
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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
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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 

Request Quote
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
 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

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
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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
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
 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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Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5. PubMed PMID: 10829079; PubMed Central PMCID: PMC18686.


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.


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Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG. Simple and highly  efficient BAC recombineering using galK selection. Nucleic Acids Res. 2005 Feb 24;33(4):e36. PubMed PMID: 15731329; PubMed Central PMCID: PMC549575.


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.


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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.


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Gregg CJ, Lajoie MJ, Napolitano MG, Mosberg JA, Goodman DB, Aach J, Isaacs FJ, Church GM. Rational optimization of tolC as a powerful dual selectable marker for genome engineering. Nucleic Acids Res. 2014 Apr;42(7):4779-90. doi: 10.1093/nar/gkt1374. Epub 2014 Jan 22. PubMed PMID: 24452804; PubMed Central PMCID: PMC3985617.

Li XT, Thomason LC, Sawitzke JA, Costantino N, Court DL. Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli. Nucleic Acids Res. 2013 Dec;41(22):e204. doi: 10.1093/nar/gkt1075. Epub 2013 Nov 6. PubMed PMID: 24203710; PubMed Central PMCID: PMC3905872.

Wang H, Bian X, Xia L, Ding X, Müller R, Zhang Y, Fu J, Stewart AF. Improved seamless mutagenesis by recombineering using ccdB for counterselection. Nucleic Acids Res. 2014 Mar;42(5):e37. doi: 10.1093/nar/gkt1339. Epub 2013 Dec 24. PubMed PMID: 24369425; PubMed Central PMCID: PMC3950717.
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