Preparation of intact yeast artificial chromosome DNA for transgenesis of mice.
Transgenesis with large DNA molecules such as yeast artificial chromosomes (YACs) has an advantage over smaller constructs in that an entire locus and all its flanking cis-regulatory elements are included. The key to obtaining animals bearing full-length transgenes is to avoid physical shearing of the DNA during purification and microinjection. This protocol details how to prepare intact YAC DNA for transgenesis of mice and involves separation of YAC DNA from yeast chromosomal DNA by pulsed field gel electrophoresis, concentration to a range suitable for microinjection by second dimension electrophoresis and enzymatic digestion of matrix-embedded YAC DNA to produce a solution that can be injected. The YAC is maintained in an agarose gel matrix to avoid damage until the final steps before microinjection. Special precautions are also taken during the microinjection protocol. Transgenesis efficiency is approximately 15%; most animals carry 1-5 copies of the desired locus. This method takes 6 d for completion.
Peterson
Kenneth R
KR
Department of Biochemistry and Molecular Biology, Mail Stop 3030, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA. kpeterson@kumc.edu
D000818:Animals; D018244:Chromosomes, Artificial, Yeast; D016521:Electrophoresis, Gel, Pulsed-Field; D015180:Electrophoresis, Gel, Two-Dimensional; D018014:Gene Transfer Techniques; D006026:Glycoside Hydrolases; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D019076:Transgenes
D006026:Glycoside Hydrolases; C019624:agarase