Developing efficient modification of genomic DNA through recombineering

2000

Challenge

Traditional genetic engineering techniques use restriction enzymes to “cut” DNAs into fragments that are joined (“pasted”) with DNA ligase to produce new recombinant DNA. Though powerful and still very much in use today, these tools are too imprecise for biomedical researchers who need technologies that allow genetic changes to be made directly in genomic DNA with high fidelity and precision.

Advance

IRP scientists led by Donald Court, Ph.D., developed an in vivo recombineering (recombination-mediated genetic engineering) technique to create DNA constructs precise to the base pair. The new method utilizes homologous recombination to incorporate short pieces of synthetic single- or double-stranded DNA into the genome.

Impact

Recombineering has drastically changed the field of molecular biology and genetics by reducing both the cost and time involved in modifying genomic DNA and enabling the generation of transgenic animal models of great complexity. This has led to widespread functional genomic studies and an understanding of how genes are expressed and regulated. 

Publications

Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. (2000). An efficient recombination system for chromosome engineering in E. coli. Proc. Nat. Acad. Sci. USA. 97(11), 5978-83.

Ellis HM, Yu D, DiTizio T, Court DL. (2001). High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Nat. Acad. Sci. USA. 98(12), 6742-6.

Copeland NG, Jenkins N, Court DL. (2001). Recombineering: A Powerful New Tool for Mouse Functional Genomics. Nature Reviews Genetics. 2(10), 769-779.

Li XT, Thomason LC, Sawitzke JA, Costantino N, Court DL. (2013). Bacterial DNA polymerases participate in oligonucleotide recombination. Mol Microbiol. 88(5), 906-20.