Molecular Biology Services
BAC Recombineering Service
Recombineering (recombination-mediated genetic engineering) is a novel and powerful method enabling fast and efficient construction of vectors for subsequent manipulation of mouse genomes. It is based on homologous recombination in E. coli using recombination proteins provided from a lambda phage. This technical breakthrough is precise and independent of the presence of restriction sites and the size of the DNA molecule to be modified. Furthermore this innovative technology is significantly less laborious than traditional DNA manipulation techniques and is therefore amendable to the higher-throughput needed for a centralized service facility. Because this technology is based on homologous recombination it is incredibly versatile and can be used to create vectors for gene knockout, conditional knockout, knockin, and modified BACs for transgenic production.
The Duke Recombineering Core is a full-service facility that begins with the design of the targeting strategy based on the needs of the investigator and finishes by providing linearized DNA ready for ES cell transfection as well as suggested Southern strategies for downstream screening of homologous recombination. The Core has generated several mouse targeting vectors, including conditional knockouts and knockins. Protocols are being established to introduce other genetic markers (green fluorescent protein and Cre recombinase) into BACS for transgenic mouse and zebrafish production.
The Duke Cancer Institute Transgenic Mouse Facility’s BAC Recombineering Core also provides CRISPR modeling services for generating transgenic mice. CRISPR (clustered regularly interspaced short palindromic repeats) is a new technology that allows genome editing of mice in a manner that is faster than standard transgenic modifications and less expensive overall. Precise alterations to disrupt target genes by insertion deletion mutations, and specific base changes are now possible for the scientific community at Duke through this new service.
Our CRISPR service includes consultation with the Investigator, design and cloning of guide sequences, designing and synthesis of repair oligos (if a specific point mutation is desired), production of single guide RNA for cytoplasmic injection into fertilized mouse embryos, rapid screening of up to 50 putative founder pups, and specific protocols for genotyping offspring.
If you are interested in molecular biology services through the BAC Recombineering Core, please contact Gary Kucera.
Lab Protocols may be found HERE