Direct Isolation of Seamless Mutant Bacterial Artificial Chromosomes

George T. Lyozin1, Yasuhiro Kosaka2, Gourab Bhattacharje2, H. Joseph Yost2, Luca Brunelli3

1 University of Nebraska and Children's Hospital Medical Center, Omaha, Nebraska, 2 Eccles Institute of Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah, 3 Department of Pediatrics (Neonatology), The University of Utah School of Medicine, Salt Lake City, Utah
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 8.6
DOI:  10.1002/cpmb.34
Online Posting Date:  April, 2017
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Abstract

Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination‐mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i.e., selectable markers, this approach is prone to false positives and is labor‐intensive because it requires the subsequent removal of the selectable marker. We created Founder Principle–driven Enrichment (FPE), a method based on the population genetics “founder principle,” to directly isolate rare mutant BACs, without any selectable marker, from liquid cultures via the polymerase chain reaction (PCR). Here, we provide a detailed description of FPE, including protocols for BAC recombineering and PCR screening. © 2017 by John Wiley & Sons, Inc.

Keywords: bacterial artificial chromosome; founder principle; markerless; rare genetic variant; recombineering; selectable marker

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Founder Principle–Driven Enrichment (FPE)
  • Support Protocol 1: Recombination‐Mediated Genetic Engineering
  • Support Protocol 2: Polymerase Chain Reaction (PCR) in Liquid Cell Cultures
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Founder Principle–Driven Enrichment (FPE)

  Materials
  • Recombineering mix ( protocol 2)
  • LB agar plates (see recipe) and LB medium (see recipe)
  • Plating medium (see recipe)
  • Chloramphenicol (Cm) stock solution (e.g., 30 mg/ml in 100% ethanol; working concentration in medium or agar, 10 to 15 µg/ml)
  • Ampicillin (Amp) stock solution (e.g., 75 mg/ml in 50% ethanol; working concentration in medium or agar, 50 to 100 µg/ml)
  • SM mod or any other isotonic solution for dilution and plating bacteria
  • 96‐well flat‐bottom microtiter plates for growing cultures with volumes >50 μl and U‐bottom or V‐bottom for growing cultures with smaller volumes
  • Repetitive Distriman (Gilson) or 1‐channel electronic pipettors with multiple dispensing option for 96‐well microtiter plates
  • Thermostatically controlled bacterial incubator
  • Bacterial colony counter (optional)
  • Sterile toothpicks or 10‐µl pipet tips
  • Additional reagents and equipment for PCR screening of recombinant cultures ( protocol 3)

Support Protocol 1: Recombination‐Mediated Genetic Engineering

  Materials
  • E. coli colonies harboring mini lambda and the BAC to be mutated (Court et al., )
  • LB medium (see recipe)
  • Chloramphenicol (Cm) stock solution (e.g., 30 mg/ml in 100% ethanol; working concentration in medium or agar, 10 to 15 µg/ml)
  • Tetracycline (Tc) stock solution (e.g., 15 mg/ml in 50% ethanol; working concentration in medium or agar, 50 to 100 µg/ml)
  • Ampicillin (Amp) stock solution (e.g., 75 mg/ml in 50% ethanol; working concentration in medium or agar, 50 to 100 µg/ml)
  • 10% (v/v) glycerol, pre‐chilled
  • TB exp medium (see recipe)
  • BAC targeting vector DNA, either single‐stranded oligonucleotides or double‐stranded DNA (see Table 8.6.1 for details about single‐stranded targeting vector DNA and steps 4 to 7 of protocol 3 as an example of double‐stranded targeting vector DNA preparation) in electroporation buffer (see recipe for electroporation buffer)
  • pUC plasmid
  • Electroporation buffer (see recipe)
  • DpnI restriction endonuclease
  • 50‐ml sterile tubes
  • Shaking incubator
  • Sterile glass flasks for growing bacteria
  • Spectrophotometer and spectrophotometer cuvettes
  • 50‐ml screw‐cap centrifuge tubes
  • Thermostatically controlled shaking water bath
  • 50‐ml glass flasks
  • Refrigerated centrifuge (e.g., Sorvall Legend X1R with 50‐ml screw cap tube adapters)
  • Vacuum aspirator
  • 17 × 100–mm culture test tubes
  • 1‐mm‐gap electroporation cuvettes
  • Electroporator: Gene PulserXcell (Bio‐Rad) or similar
  • 1.5‐ and 0.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for plasmid DNA extraction (unit 1.6; Engebrecht, Brent, & Kaderbhai, ), determining DNA concentration ( appendix 3D; Gallagher, ), PCR screening of recombinant cultures ( protocol 3), and agarose gel electrophoresis (Voytas, )

Support Protocol 2: Polymerase Chain Reaction (PCR) in Liquid Cell Cultures

  Materials
  • PCR mix (see recipe)
  • Saturated DDC ( protocol 1Basic Protocol)
  • Q5 polymerase mix (NEB)
  • f and r 30ntCat→Bla primers (Table 8.6.1)
  • Plasmid harboring bla gene, e.g., pUC (Thermo Fisher Scientific)
  • 10‐ and 100‐ to 200‐µl manual and/or electronic multichannel pipettors for mixing and sampling bacterial cultures and loading agarose gels
  • Reliable 10‐µl multichannel pipet tips such as OneTouch tips (Sorenson BioScience, cat. no. 10300).
  • PCR plates
  • PCR plate sealers: reusable Corning Thermowell Sealing Mats (cat. no. 6555) or Axygen AxyMats (cat. no. AM‐96‐PCR‐RD): unlike sealing films, mats have plugs extending into each PCR plate well; because of this, they seal wells both inside and outside—plugs of the suggested mats are of different sizes for plates with corresponding internal well diameter
  • Automated thermal cycler accepting 96‐well PCR plates and allowing thermal gradients for PCR condition optimization
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Figures

Videos

Literature Cited

Literature Cited
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