Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site
© The Author(s) 2017
Received: 15 June 2017
Accepted: 17 July 2017
Published: 28 July 2017
Human genomic libraries constructed in bacterial artificial chromosome vectors were utilized to make physical maps of all 23-chromosome pairs and as the templates for DNA sequencing to aid in the completion of the Human Genome Project. The goal of this study was to modify the BAC vector pBeloBAC11 so that genomic inserts contained in this vector could be subjected to bidirectional transposon-mediated nested deletions from the wild-type and mutant loxP sites present.
An oligonucleotide containing a mutant loxP 2272 site and a XhoI restriction enzyme sequence was designed and inserted at the SfiI restriction site located approximately 200 basepairs upstream of the lacZ gene in pBeloBAC11. Clones containing the desired insert were identified by XhoI restriction digests since an additional band was generated. This transposon-mediated deletion technology allows researchers to identify the boundaries of cis-acting elements and genes.
Bacterial artificial chromosome (BAC) and P1 artificial chromosome (PAC) vectors were used to generate human genomic libraries for the Human Genome Project (HGP) since they could accommodate inserts up to 300 kilobases (kb) and did not generate chimeric clones [1, 2]. Since the PAC vectors were derived from the plasmid pAd10sacBII, they all contained a loxP site that is part of the bacteriophage P1 genome [3, 4].
A mini-Tn10 transposon plasmid containing a loxP site was developed to retrofit P1 clones to generate nested deletions . We then used this technology to demonstrate that large nested deletions could be generated in both PAC and BAC clones . A study by Lee and Saito  investigated the role that each nucleotide in this 34-bp sequence plays in the recombination process. They identified two double-base substitutions in the 8-bp spacer region (5171 and 2272) that efficiently recombined with an identical mutant but not with the other mutant or the wild-type loxP site.
The goal of this study was to modify the BAC cloning vector pBeloBac11 to make it more versatile for a variety of studies. Oligonucleotides containing the 2272 version of the mutant loxP site and an internal XhoI restriction sequence were designed and ligated into the SfiI site of this BAC vector. Researchers can transform transposon plasmids carrying either a wild type or mutant loxP site into the E. coli cells containing any BAC clone of interest created in pBeloBac11 to generate transposon-mediated nested deletions from both ends of the genomic DNA.
The following oligonucleotides were synthesized and gel purified (Sigma-Genosis): 5′CTCGAGATAACTTCGTATAAAGTATCCTATACGAAGTTATCCC3′ and 5′ATAAACTTCGTATAGGATACTTTATACGAAGTTATCTCGAGGGG3′. Each oligonucleotide was dissolved in 10 mM Tris–Cl, 1 mM EDTA, pH 8.0 (TE) at a final concentration of 1 μg/ml and incubated at 4 °C overnight. The oligonucleotides were combined in the presence of 50 mM NaCl and heated to 80 °C for 5 min and then slowly cooled to room temperature.
A 500-fold molar excess of the double-stranded oligonucleotide was incubated with pBeloBac11 DNA that had been digested with SfiI for 2 h at 37 °C (New England BioLabs) in the presence of 1 U of T4 DNA ligase (Invitrogen) at 15 °C overnight. The ligation reaction was heat inactivated at 70 °C for 15 min and then spot dialyzed against 0.5X TE for 1 h. Two μl aliquots of the ligation reaction were mixed with 25 μl of electrocompetent DH10B cells (Invitrogen), placed into a 1 cm gap cuvette (BTX) and electroporated at 2.5 kV and 129 Ohms using the BTX Electro Cell Manipulator 600. The cells were allowed to recover in 1 ml SOC media (Invitrogen) at 37 °C and 225 rpm for 1 h and then 100 μl aliquots were spread onto LB + 34 μg/ml CM (Sigma) plates. Individual colonies were grown overnight in 2.5 ml of LB + CM media and DNA was generated using an alkaline lysis miniprep procedure . The DNA samples were digested with 10 U of XhoI for 2 h at 37 °C (New England BioLabs) and electrophoresed in a 0.8% agarose gel in 0.5X TBE.
pBeloBAC11 loxP* cannot be propagated in mammalian cells.
The author declares that he has no competing interests.
Availability of data and materials
pBeloBac11-loxP* is available at Addgene (ID# 60342).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Shizuya H, Birren B, Kim U, Mancino V, Slepak T, Tachiri Y, Simon M. Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci USA. 1992;89:8794–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Ioannou PA, Amemiya CT, Garnes J, Kroisel PM, Shizuya H, Chen C, Batzer MA, de Jong PJ. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nat Genet. 1994;6:84–9.View ArticlePubMedGoogle Scholar
- Pierce JC, Sauer B, Sternberg N. A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficiency. Proc Natl Acad Sci USA. 1992;89:2056–60.View ArticlePubMedPubMed CentralGoogle Scholar
- Shizuya H, Kouros-Mehr H. The development and applications of the bacterial artificial chromosome system. J Med. 2001;50:26–30.Google Scholar
- Chatterjee PK, Sternberg N. Retrofitting high molecular weight DNA cloned in P1: introduction of reporter genes, markers selectable in mammalian cells and generation of nested deletions. Genet Anal. 1996;13:33–42.View ArticlePubMedGoogle Scholar
- Chatterjee PK, Coren JS. Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed loxP site. Nucleic Acids Res. 1997;25:2205–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee G, Saito I. Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene. 1988;216:55–65.View ArticleGoogle Scholar
- Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979;7:1513–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Coren JS, Sternberg N. Construction of a PAC vector system for the propagation of genomic DNA in bacterial and mammalian cells and subsequent generation of nested deletions in individual library members. Gene. 2001;264:11–8.View ArticlePubMedGoogle Scholar