Recombineering‐Based Procedure for Creating BAC Transgene Constructs for Animals and Cell Lines

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The use of BAC/P1 as a vector for the generation of a transgene has gained popularity after the genomic annotation of many organisms was completed (often based on the respective BAC library). Large?scale generation of BAC transgenic mice has proven that BAC transgene approaches have less integration position effects and dosage artifacts when compared with traditional transgenic approaches. Also, a BAC can achieve the same tissue?specific expression as a knock?in of the same gene with less effort and shorter time of establishment. The ??RED recombinogenic system has been used to manipulate DNA constructs with site?directed mutagenesis, truncation, and tagging with an epitope tag or as a fusion protein by homologous recombination, as well as used here to modify many BACs with various transgenes. The recombineering plasmid, pKD46, is used to fabricate BAC transgenic constructs that can be used in generating transgenic organisms as well as used in mammalian cell culture. Curr. Protoc. Mol. Biol. 95:23.14.1?23.14.28. © 2011 by John Wiley & Sons, Inc.

Keywords: BAC; recombineering; transgene

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Introduction
Basic Protocol 1: Fabrication of BAC Transgene Construct Through Recombineering
Support Protocol 1: Purify Targeting Cassette with a Safeblue Gel
Support Protocol 2: Prepare Electrocompetent Cells
Support Protocol 3: Electroporation of a Plasmid into DH10B and its Derivatives
Support Protocol 4: Storing and Handling Recombinogenic Bacteria Strains
Support Protocol 5: Acquisition and Confirmation of BACs
Support Protocol 6: Obtain BAC DNA for Transformation
Alternate Protocol 1: Assemble a Targeting Cassette by PCR with Long Primers
Alternate Protocol 2: Overlapping PCR
Alternate Protocol 3: Arm Extension on the Targeting Cassette
Alternate Protocol 4: Sequential Targeting for Transgene Swapping
Alternate Protocol 5: Selection Marker Removal with Titration of Recombinase
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Fabrication of BAC Transgene Construct Through Recombineering

Materials

Bacteria containing BAC
Low‐salt LB medium (unit 1.1 )
20 µg/ml chloramphenicol (34 mg/ml stock in ethanol; Sigma)
pKD46 and its derivatives (Dr. B. Wanner)
Electrocompetent cells (see protocol 3 )
SOC medium (NEB)
Agar plates (unit 1.1 )
100 µg/ml ampicillin (50 mg/ml stock in water; Sigma)
Phusion high‐fidelity DNA polymerase (Finnzymes) or similar high‐fidelity enzyme
pGEM‐T‐Easy (Promega)
E. coli competent cells for cloning: DH10B (Invitrogen), NEB 10‐beta (NEB), Stable 2 or 4 (stb2/4, Invitrogen)
Isopropylthio‐β‐D‐galactoside (IPTG; 1 M, 20 µl/plate)
5‐Bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside (X‐gal, 50 mg/ml in dimethylformamide, 50 µl/plate)
Restriction endonucleases (NEB)
Antibiotics:
- 25 µg/ml kanamycin (50 mg/ml stock in water; Sigma)
- 25 µg/ml zeocin (100 mg/ml stock in water; Invitrogen)
- 50 µg/ml hygromycin (100 mg/ml stock in water; Invivogen)
Gel extraction kit (Qiaquick, Qiagen)
0.1% L‐Arabinose (10% stock in water; Sigma)

32°C orbital shaker incubators
Spectrophotometer with 1‐ml disposable plastic cuvettes
1.5‐ and 2‐ml microcentrifuge tubes
Electroporator (Eppendorf Electroporator 2510) with 1‐mm electroporation cuvettes
2‐ml centrifuge tubes
Bench‐top centrifuge with temperature control (Eppendorf 5810R)
50‐ml culture tubes
Spectrophotometer (Nanodrop ND1000) with 1‐ml disposable plastic cuvettes (ThermoSpectronic‐Biomate 3)
96‐well assay plates
14‐ml centrifuge tubes

Additional reagents and equipment for PCR (unit 15.1 ), agarose gel electrophoresis (unit 2.5 ), DNA preparation and restriction endonuclease digestion (unit 3.1 ), and pulsed‐field gel electrophoresis (unit 2.5 )

Support Protocol 1: Purify Targeting Cassette with a Safeblue Gel

Materials

Safeblue dye (SYBR safe, Invitrogen)
DNA sample
Gel purification kit (Qiagen)

Safe Imager blue‐light transilluminator (Invitrogen)
Scalpel or razor blade
NanoDrop

Additional reagents and equipment for agarose gel electrophoresis (unit 2.5 )

Support Protocol 2: Prepare Electrocompetent Cells

Materials

High‐density culture (culture grown to saturation) of appropriate cells, e.g., DH10B (Invitrogen) or Stable 2 or 4 (stb2/4, Invitrogen)
10% glycerol

Spectrophotometer
2‐ml microcentrifuge tubes
50‐ml centrifuge tubes
Refrigerated centrifuge

Support Protocol 3: Electroporation of a Plasmid into DH10B and its Derivatives

Materials

Resuspended electrocompetent cells (DH10B or its derived cells, DY380, EL250, EL350; see protocol 3 )
Freshly prepared DNA: BAC (1/10 miniprep), plasmid (50 ng) or targeting cassette (100∼200 ng)
SOC medium (NEB)
LB agar plate containing appropriate antibiotics for selection

Electroporator (Eppendorf electroporator 2510) with 1‐mm electroporation cuvettes
1.5‐ml microcentrifuge tubes
32°C (37°C for non‐recombinogenic bacteria) orbital shaking incubator

Support Protocol 4: Storing and Handling Recombinogenic Bacteria Strains

Materials

Recombinogenic bacteria
LB medium
Glycerol (or DMSO) to make final 25% (or 7%)

5‐ml transparent centrifuge tubes
32° and 42°C incubator

Support Protocol 5: Acquisition and Confirmation of BACs

Materials

LB medium with 20 µg/ml chloramphenicol
BAC‐containing cells in glycerol stock or grown on agar
50 mM Tris⋅Cl (pH 8.0)/10 mM EDTA/100 µg/ml RNase A
Miniprep kit (Qiagen), optional
Isopropanol
70% ethanol
10 mM Tris⋅Cl buffer, pH 8.5 ( appendix 22 )

14‐ml conical tubes
32°C orbital shaking incubator
2‐ml centrifuge tubes
Tabletop centrifuge
1.5‐ml microcentrifuge
1‐ml pipets

Support Protocol 6: Obtain BAC DNA for Transformation

Materials

Plasmid DNA
Dpn I enzyme
HFPCR‐amplified transgene with selection marker
Phusion DNA polymerase (Finnzymes) or similar high‐fidelity enzyme
PCR primers
PCR nucleotide mix
Thermal cycler

Alternate Protocol 1: Assemble a Targeting Cassette by PCR with Long Primers

Materials

Targeting cassette with the 50‐mer homologous arms (see protocol 8 ) generated using two sets of primers: transgene primers set or 50 mer+transgene primer set
Primer L/R (can be same as overlapping PCR primer pairs or ID primers)
Taq polymerase (NEB)
pGEM‐T‐Easy (Promega)
Agar plate with 100 µg/ml ampicillin as well as X‐Gal and IPTG
LB medium
Miniprep kit (Qiagen)
pKD46 and its derivatives
Recombinogenic bacteria (DY380/SW102, EL250/SW105. EL350/SW106) (Dr. N. Copeland)

Thermal cycler
Electroporator and cuvettes

Alternate Protocol 2: Overlapping PCR

Materials

Targeting cassette with 50‐mer homologous arms (see protocol 8 ) that is generated by two sets of primers: transgene primer set and 50 mer+transgene primer set or targeted BAC with shared components (such as eGFP‐pA BAC)
Larger transgene with shared components (second TC), preferably with a different selection marker then the first targeting cassette
Recombinogenic plasmid: pKD46
Genotyping primer‐sets that can distinguish first targeting and second targeting product

Alternate Protocol 3: Arm Extension on the Targeting Cassette

Materials

BAC‐tg
EL250 or EL350 cells
Agar plates with proper selection
LB medium
Arabinose

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Figures

Figure 23.14.1 Insertion site of a transgene. The transcription start site is located at the beginning of the 5′UTR (untranslated region); all translated regions (coding sequence for protein‐CDS‐from start codon, ATG, to stop codon) are black. Inside the 3′UTR, the IRES‐CDS should be located before the poly‐A sites.

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Figure 23.14.2 Flow chart of BAC targeting with targeting cassette. On the targeting cassette the left arm of homology (LAH) and right arm of homology (RAH) add a homologous region to the targeting cassette that matches the left and right flanking portion of the targeted site on the BAC. The genotyping primers (GT) must be located outside of the arms of homology. If the selection marker is flanked by FRT sites then use EL250 to remove the selection marker. If flanked by LoxP sites, use EL350.

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Literature Cited

	Copeland, N.G., Jenkins, N.A., and Court, D.L. 2001. Recombineering: A powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2:769‐779.
	Datsenko, K.A. and Wanner, B.L. 2000. One‐step inactivation of chromosomal genes in Escherichia coli K‐12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97:6640‐6645.
	Gong, S., Zheng, C., Doughty, M.L., Losos, K., Didkovsky, N., Schambra, U.B., Nowak, N.J., Joyner, A., Leblanc, G., Hatten, M.E., and Heintz, N. 2003. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917‐925.
	Lee, E.C., Yu, D., Martinez de Velasco, J., Tessarollo, L., Swing, D.A., Court, D.L., Jenkins, N.A., and Copeland, N.G. 2001. A highly efficient Escherichia coli‐based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56‐65.
	Poser, I., Sarov, M., Hutchins, J.R., Hériché, J.K., Toyoda, Y., Pozniakovsky, A., Weigl, D., Nitzsche, A., Hegemann, B., Bird, A.W., Pelletier, L., Kittler, R., Hua, S., Naumann, R., Augsburg, M., Sykora, M.M., Hofemeister, H., Zhang, Y., Nasmyth, K., White, K.P., Dietzel, S., Mechtler, K., Durbin, R., Stewart, A.F., Peters, J.M., Buchholz, F., Hyman, A.A. and 2008. BAC TransgeneOmics: A high‐throughput method for exploration of protein function in mammals. Nat. Methods 5:409‐415.
	Wagner, M. and Koszinowski, U.H. 2004. Mutagenesis of viral BACs with linear PCR fragments (ET recombination). Methods Mol. Biol. 256:257‐268.
	Yang, X.W., Model, P., and Heintz, N. 1997. Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol. 15:859‐865.
	Zhang, Y., Buchholz, F., Muyrers, J.P., and Stewart, A.F. 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20:123‐128.
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