BAC Recombineering Fundamentals
BAC recombineering utilizes homologous recombination between short homologous sequences (typically 50 base pairs minimum) in E. coli strains expressing bacteriophage lambda recombination genes and Cre recombinase.
BAC Source Selection and Characterization
Research teams typically identify appropriate BAC clones from existing genomic libraries covering mouse, human, rat, or other organism genomes. Clone selection requires careful attention to insert structure to ensure BAC spans the complete genomic region of interest including all relevant regulatory elements.
Recombineering Cassette Insertion
Standard BAC recombineering procedures insert selection cassettes and homologous recombination arm sequences into BAC clones using FRT flanked cassettes for subsequent removal.
Large Genomic Fragment Preservation
BAC targeting excels for applications requiring preservation of complete regulatory elements controlling endogenous gene expression.
Copy Number and Integration Site Considerations
BAC inserts at safe harbor loci (H11, ROSA26, HPRT) typically integrate as single copy events when ES cell characterization confirms appropriate clones.
Internal Rearrangement Risk Assessment
Large genomic fragments carry inherent risk of internal rearrangement during bacterial propagation, recombineering procedures, or ES cell targeting. Careful strain selection using recombineering proficient strains minimizes risk.
ES Cell Targeting of Large Recombineered BACs
Targeting vector construction using recombineered BACs creates very large targeting constructs (often 200+ kilobases) requiring special electroporation and ES cell selection protocols.
Pre Germline Characterization Importance
Pre germline ES cell analysis becomes particularly critical for large insert targeting to detect internal rearrangement events originating during vector construction or ES cell targeting.
Junction Characterization
PCR and sequencing across insertion junctions between endogenous genome and transgene sequences confirms correct targeting at intended locus.
Timeline and Project Planning for BAC Targeting
BAC targeting projects involve complex allele design and verification. Contact us for current timeline estimates based on your specific project requirements.
Phase 1: BAC Selection and Characterization
Genomic library searches identify candidate BAC clones. Physical characterization via restriction mapping and gel electrophoresis confirms insert structure and boundaries.
Phase 2: Recombineering Vector Construction
Targeting vector sequences and functional cassettes insert into BAC clones via sequential recombineering rounds. Each round requires bacterial growth, selection, and characterization.
Phase 3: ES Cell Targeting and Selection
Targeting vectors transfect into ES cells, undergo selection, and yield correctly targeted clones at frequencies 10 to 100 fold lower than standard vectors.
Phase 4: Chimera Generation
Correctly targeted ES cell clones contribute to chimeric mice via blastocyst injection. Germline transmission verification through breeding establishes stable transgenic lines.
Applications Ideally Suited to BAC Targeting
Disease Modeling with Large Human Genomic Inserts
Researchers can insert disease associated human loci (100+ kilobases including regulatory elements) into mouse genomes while maintaining human genetic context relevant to disease mechanisms.
Regulatory Element Studies
Studies examining how multiple enhancers and silencers coordinate gene expression require BAC insert capacity to preserve these relationships in transgenic contexts.
Complex Transgenic Studies
Combining multiple functional elements (promoters, genes, reporters, regulatory cassettes) in defined spatial arrangements benefits from BAC recombineering flexibility.
Publications Utilizing BAC Targeting
Jiang Y, Sachdeva K, Goulbourne CN, Berg MJ, Peddy J, Stavrides PH, Pensalfini A, Pawlik M, Malampati S, Whyte L, Basavarajappa BS, Shivakumar S, Bleiwas C, Smiley JF, Mathews PM, Nixon RA. 2025. Increased neuronal expression of the early endosomal adaptor APPL1 leads to endosomal and synaptic dysfunction with cholinergic neurodegeneration J Neurosci 29(45): e2331242025 ↗
Serrano J, Boyd J, Brown IS, Mason C, Smith KR, Karolyi K, Maurya SK, Meshram NN, Serna V, Link GM, Gardell SJ, Kyriazis GA. 2024. The TAS1R2 G-protein-coupled receptor is an ambient glucose sensor in skeletal muscle that regulates NAD homeostasis and mitochondrial capacity Nat Commun 15(1): 4915 ↗
What Researchers Say
“The Hephaestin flox model ingenious has made for us has been great. It has helped generate eight research publications.”
— Joshua Dunaief, PhD, MD
University of Pennsylvania
Start Your BAC Large Scale Targeting Project
BAC recombineering enables preservation of complex regulatory elements and large genomic features critical for physiologically relevant transgenic models. Our experienced team can guide BAC selection, manage recombineering construction, oversee ES cell targeting, and deliver study ready mice efficiently.