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BAC transgenics
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BAC transgenic

BAC transgenic

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 BAC transgenic mouse model  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

 C57BL/6 -  BAC transgene pronuclear microinjection
MBAC001
C57BL/6 strain BAC transgene pronuclear microinjection


(includes BAC purification and founder genotyping)
3 - 4 months
2 BAC founders
Custom mouse strain -  BAC transgene pronuclear microinjection
MBAC002
129, Balb/c, FVB/N, NOD


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)

MBAC003

 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

MBAC004

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

MBAC005

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
MBAC006
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
MBAC007
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 integretated 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 indidvidual BAC founder lines.
 genotyping protocol

 Mouse colony husbandry & management

MBAC008

 mouse colony management including breeding, weaning, sampling, monitoring, per cage per day for the production of founders and F-generation pups

references
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Murphy KC. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol. 1998 Apr;180(8):2063-71. PubMed PMID: 9555887; PubMed Central PMCID: PMC107131.


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.


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.


Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A. 2000 May 23;97(11):5978-83. PubMed PMID: 10811905; PubMed Central PMCID: PMC18544.


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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Testa G, Zhang Y, Vintersten K, Benes V, Pijnappel WW, Chambers I, Smith AJ, Smith AG, Stewart AF. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat Biotechnol. 2003 Apr;21(4):443-7. Epub 2003 Mar 10. PubMed PMID: 12627172.


Poser I, Sarov M, Hutchins JR, Hériché JK, Toyoda Y, Pozniakovsky A, Weigl D,  Nitzsche A, Hegemann B, Bird AW, Pelletier L, Kittler R, Hua S, Naumann R, Augsburg M, Sykora MM, Hofemeister H, Zhang Y, Nasmyth K, White KP, Dietzel S, Mechtler K, Durbin R, Stewart AF, Peters JM, Buchholz F, Hyman AA. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat Methods. 2008 May;5(5):409-15. doi: 10.1038/nmeth.1199. Epub 2008 Apr 6. Erratum in: Nat Methods. 2008 Aug;5(8):748. PubMed PMID: 18391959; PubMed Central PMCID: PMC2871289.


Ohtsuka M, Kimura M, Tanaka M, Inoko H. Recombinant DNA technologies for construction of precisely designed transgene constructs. Curr Pharm Biotechnol. 2009 Feb;10(2):244-51. Review. PubMed PMID: 19199958.


Rogatcheva MM, Rund LA, Swanson KS, Marron BM, Beever JE, Counter CM, Schook LB. Creating porcine biomedical models through recombineering. Comp Funct Genomics. 2004;5(3):262-7. doi: 10.1002/cfg.404. PubMed PMID: 18629152; PubMed Central PMCID: PMC2447442.


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.


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.


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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.

Rondelet A, Pozniakovsky A, Leuschner M, Poser I, Ssykor A, Berlitz J, Schmidt N,  Hyman AA, Bird AW. ESI mutagenesis: A one-step method for introducing point  mutations into bacterial artificial chromosome transgenes. bioRiv. doi: 10.1101/844282 Epub 2020 Apr 23. https://www.biorxiv.org/content/10.1101/844282v2

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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