Successful use of HTF as a basal fertilization medium during SEcuRe mouse in vitro fertilization.

OBJECTIVE: The ever-increasing number of genetically engineered mouse models highlights the need for efficient archiving and distribution of these lines. Sperm cryopreservation has become the preferred technique for the majority of these models due to its low requirement of costs, time, and experimental animals. Yet, current in vitro fertilization (IVF) protocols either exhibit decreased fertilization efficiency for the most popular C57BL/6 strain, as recently demonstrated by us, or require costly and difficult-to-prepare media, respectively. As a result, we previously developed SEcuRe, a modified IVF protocol with low costs and high fertilization efficiency. The popular basal fertilization medium, Cook's® proprietary "Research vitro fert" (RVF), used in this protocol has recently been discontinued. As a result, the application of the SEcuRe approach and other IVF protocols employing this medium has been severely limited. RESULTS: Here we show that human tubal fluid (HTF), a popular and widely available medium with a known composition, can be used as a basal fertilization medium instead of RVF. Comparison of RVF and HTF during 58 independent SEcuRe IVFs with cryopreserved C57BL/6 sperm revealed equal fertilization and live birth rates. In addition, we demonstrate that HTF has a substantially extended shelf-life by utilizing commercial HTF that was six months past its expiration date, yet did not affect fertilization during IVF or subsequent embryo development. This finding not only increases the economic value of our modified method, but also validates it once more. Our results demonstrate that common, shelf-life extended HTF can be used in SEcuRe IVF in place of now-discontinued RVF medium and ensure the applicability of the method, which we since termed SEcuRe 2.0. Our modified SEcuRe 2.0 strategy will assist researchers to efficiently archive and distribute genetically engineered mouse models in a cost-effective, easily adaptable, and 3R-compliant manner with minimal animal use.

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes.

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.