Clone and verify the transgene. Use unique restriction sites at the ends of the construct for vector removal as prokaryotic vector sequences often interfere with the expression. Unique markers such as reporter genes such as GFP, LacZ, non-mouse proteins or epitope tag are necessary to assay the expression and distinguish it from the endogenous gene. Sequence the junction fragments to confirm the presence of a functional promoter, initiation codon and polyadenylation signal. If at all possible, test the transgene for expression in a tissue culture system before attempting the generation of transgenic mice. GTF can perform transgene expression experiments for an additional cost.
Establish a screening method and provide the evidence that you have a PCR or Southern blot assay that detects the transgene, ideally, at a single copy concentration. A second assay that will detect an endogenous mouse gene (e.g. beta-globin, ActB, GAPDH) will demonstrate that the DNA preparations are suitable for PCR. When animals are tested with both assays, no transgenic founder is mistakenly discarded as false negative. If Southern blot assay is needed to determine the copy number, integration site number, and transgene integrity in the transgenic founders prior to breeding, please see more information on the preparation of copy standards.
The quality of DNA preparation is the most likely critical factor affecting the efficiency of transgenic mouse production. DNA should be free of any contaminant and at the appropriate concentration. Perform a restriction enzyme digest on the cloning vector to separate 20µg of the transgene insert from the cloning vector. Run out a few hundred nanograms on a minigel to determine that the digest is complete and that the bands are of the correct size. TGF can perform a restriction enzyme digest and purify the transgene if required.
Prepare 50-100 ng/µl in at least 100µl volume of sterile 0.2µm filtered water. It is strongly recommended to use NanoDrop to quantify and assess the purity of the final DNA preparation BEFORE submitting the service request. Please, provide NanoDrop results to GTF staff.