Conventional DNA injection-based methods are successful in generating transgenic animals and have remained nearly unchanged over the last few decades. Lentiviral vectors are alternative powerful tool for generating transgenic animals, in part because of their ability to incorporate into genomic DNA with high efficiency. This chapter describes lentiviral vectors used to generate transgenic mice and rats. We discuss the protocols and methods in high enough detail such that researchers who are accustomed to creating transgenic animals by pronuclear injection can smoothly transition to using lentiviral transgenesis. We will briefly outline the general principle of the lentiviral expression system and focus specifically on the methods used to generate lentiviral vectors, produce lentiviral particles, inject lentivirus into the fertilized oocytes, and transplant them into the pseudopregnant females. In addition to the surgical aspects of the experiment, we will describe methods to produce high titer lentivirus. Finally, we will discuss the limitations of lentiviral transgenesis and summarize information that will be useful for troubleshooting.
Key wordsLentivirus vector Transgenic mouse Transgenic rat Lentiviral transgenesis Microinjection shRNA Protocol
T.N. is supported by John Merck Fund, Hellman Foundation, and NARSAD. C.C.H. is supported by The Prinses Beatrix Fonds, Netherlands Organization for Scientific Research (NWO-ALW and NWO-CW-ECHO), Netherlands Organization for Health Research and Development (ZonMw-VIDI, ZonMw-TOP), Human Frontier Science Program Career Development Award (HFSP-CDA), and European Science Foundation (European Young Investigators (EURYI) Award).
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