This Facility uses recombineering (genetic engineering by recombination) to make DNA constructs and targeting vectors that can then be used to generate genetically modified animals. Using BACs as starting material, which are readily available for virtually every gene in the genome and can be identified from Internet-accessible public databases, core can generate knock-in, knock-out and reporter gene constructs for use in transgenic animals. Furthermore, BACs are 100-200 kb in size, making them large enough to cover an entire gene to direct the expression of transgene as that of replaced endogenous gene.
Our Facility has been in operation for more than 10 years and has made many complex BAC-derived constructs for use in mice. For example, we modified BACs to express epitope tagged proteins, EGFP (Enhanced Green Fluorescence Protein), hPLAP (human Placental Alkaline Phosphatase) and beta-galactosidase in genetically-defined cell types. We have also generated constructs expressing Cre recombinase, Tet on/off trans-activators and optogenetic proteins to study neural circuit function. We can also use recombineering to generate various mutations at the gene level including: exon swaps, duplications, truncations, deletions, point mutations and conditional gene modifications. Combined with artificial endonuclease such as zinc-finger or TALE nuclease, it is also possible to perform chromosome engineering.
BAC Core staff work closely with clients and with the Animal Model Core Facility at UNC to generate transgenic, gene-targeting, conditional mutagenesis animals. We provide free initial consultation in the design of transgenic or gene targeting constructs as well as the continue support of making construct, screening ES cells and establishing mutant mouse lines.
Additional supports for the technique of Molecular Biology include DNA library construction, making viral vector (especially rAAV) and in situ hybridization. In situ hybridization remains a critical tool for neuroscience research. It is the most efficient method to verify results of gene chip studies related to particular brain regions. We adapted both whole embryos and sections from a variety of embryonic, early postnatal, and adult mice. Other models animal such a whole zebra fish embryos, chick embryos, human tissues, or sections through developmental and adult rat brain have been used.
- To generate transgenic and gene-targeting constructs using recombineering with Bacterial Artificial Chromosome (BAC).
- Targeting mutagenesis of point mutation, truncation in plasmid as large as a BAC.
- To assist neuroscientists in generating knockin/out mice by screening gene targeted embryonic stem (ES) cells (includes 5’ and 3’ probe generation and Southern blot screening).
- To generate expression and recombinant adenoviral (rAAV) plasmid constructs.
- To prepare massively parallel (next-generation) sequencing libraries (RNA-seq, ChIP-seq).
- In situ hybridization with RNA probes on tissue sections or as whole mount.