Year: 2025
Type: preprint
Abstract: The purpose of this protocol is to describe the identification of transgenes in genomic mouse DNA. The use of copy number standards allows one to determine the sensitivity of the genotyping assay. This is important because transgenic founders or CRISPR founders are often mosaic and may carry less than one genomic copy of the inserted DNA when tail tip or other genomic DNA is assayed. PCR screens must be designed to detect transgene DNA at 0.1 copy level or lower. Insensitive assays are known to be the reason that transgenic founders were not detected on numerous occasions. To avoid this experimental artifact PCR assays need to be standardized copy standard template prepared by mixing non-transgenic tail DNA with a known amount of transgene DNA. When sensitive and specific primers are used then the occurrence of false negative results during genotyping can be eliminated.If primers do not meet sensitivity requirements or if they produce more than one band on a gel with copy standards then new primer pairs are ordered and tested until a suitable pair is identified.
Authors: Thom Saunders, Honglai Zhang, Jennifer Leo, Sivakumar Jeyaraja, Eden A. Dulka, Zachary T. Freeman
Institution: University of Michigan
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4414328651