A rapid and effective nonsurgical artificial insemination protocol using the NSET™ device for sperm transfer in mice without anesthesia.

Artificial insemination (AI) is an assisted reproductive technique that is implemented successfully in humans as a fertility treatment, performed extensively for commercial breeding of livestock, and is also successful in laboratory rodents. AI in the mouse may be especially useful for breeding of transgenic or mutant mice with fertility problems, expansion of mouse colonies, and as an alternative to in vitro fertilization. Nonsurgical AI techniques for the mouse have been described previously but are not often implemented due to technical difficulties. Here we compare various protocols for preparation of CD1 recipients prior to AI for naïve (in estrus), ovulation-induced, and superovulated females. Timing of hormone administration relative to sperm delivery is also compared. An improved protocol for nonsurgical AI in mice is described, which incorporates a convenient hormone administration schedule for female recipients and rapid, non-stressful sperm transfer without the need for anesthesia or analgesia.

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination.

For successfully maintaining pregnancy with embryo transfer or artificial insemination, female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

Rat Embryonic Stem Cell Transgenesis.

The availability of reliable germline competent rat embryonic stem cell (ESC) lines that can be genetically manipulated provides an important tool for generating new rat models. Here we describe the process for culturing rat ESCs, microinjecting the ESCs into rat blastocysts, and transferring the embryos to surrogate dams by either surgical or non-surgical embryo transfer techniques to produce chimeric animals with the potential to pass on the genetic modification to their offspring.

Nonsurgical Embryo Transfer Protocol for Use with the NSET™ Device.

Genetically modified embryos must be transferred to a suitable female recipient for development to pups. Nonsurgical embryo transfer is a fast and efficient method used to deliver blastocyst stage embryos to the uterine horn of recipient females. The efficiency of recovery of pups after nonsurgical embryo transfer is similar to the efficiency after surgical transfer. However, nonsurgical transfer eliminates the pain and distress caused by the surgical procedure and provides a refinement in accordance with Russel and Burch's "3Rs" (The principles of humane experimental technique. Methuen & Co., London, 1959), an ethical framework for animal research. This method is also useful for rederivation of mouse strains. Rederivation is important for either removal of potential pathogens from an incoming mouse strain after shipping, or within a facility to obtain a clean mouse colony.

Media optimization to promote rat embryonic development to the blastocyst stage in vitro.

Efficient model production in rats that incorporates newly developed genetic editing and embryo transfer tools, such as CRISPR/Cas9 technology and non-surgical embryo transfer, requires availability of an optimal embryo culture system. However, current technologies for in vitro manipulation of rat gametes, including embryo culture techniques, are less advanced compared to those in mice. In this study, we (1) identified a culture medium that was able to support optimal rat embryonic development by comparing two rat culture media: mR1ECM (modified rat 1-cell embryo culture medium) and KSOM-R (modified potassium simplex optimized medium for rats), and (2) evaluated the effect of glutamine dipeptides: alanyl-l-glutamine and glycyl-l-glutamine, on rat embryonic development. We also investigated the possibility of simplifying the KSOM-R culture procedure by increasing the volume of culture medium, reducing the need for daily medium changes. The results showed that rat embryos cultured in KSOM-R developed faster than those cultured in mR1ECM. Both alanyl-l-glutamine and glycyl-l-glutamine showed detrimental effects on rat embryonic development when supplemented in KSOM-R at the same concentration as glutamine. By increasing the volume of KSOM-R, rat zygotes were able to develop without daily medium refreshment at a similar rate and developmental competence as those in smaller volumes with daily medium changes. These results represent important improvements to rat embryo culture methods and will assist in more efficient production of rat models.

A Nonsurgical Embryo Transfer Technique for Fresh and Cultured Blastocysts in Rats.

The use of a nonsurgical embryo transfer technique in rodents eliminates the potential pain, distress, and health complications that may result from a surgical procedure and as such, represents a refinement in rodent assisted reproductive techniques. A nonsurgical technique has not been previously developed for use with rat embryos. Here we describe an efficient method to deliver either fresh or cultured blastocyst stage embryos to the uterine horn of pseudopregnant female rats using a rat nonsurgical embryo transfer (rNSET) device. The rNSET device is composed of a Teflon catheter and a hub that attaches to a 2 µL pipette. Oxytocin is used to dilate the cervix before the delivery of blastocysts, allowing passage of the rNSET catheter directly into the uterine horn for embryo delivery. The efficiency of recovery of pups after nonsurgical embryo transfer is similar to the efficiency after surgical embryo transfer. Furthermore, the technique is not stressful to the subjects, as demonstrated by the absence of a decrease in weight or increase in fecal corticosterone level in recipients of embryos delivered nonsurgically, without the use of anesthesia or analgesia.

Synthetic Biology-derived triterpenes as efficacious immunomodulating adjuvants.

The triterpene oil squalene is an essential component of nanoemulsion vaccine adjuvants. It is most notably in the MF59 adjuvant, a component in some seasonal influenza vaccines, in stockpiled, emulsion-based adjuvanted pandemic influenza vaccines, and with demonstrated efficacy for vaccines to other pandemic viruses, such as SARS-CoV-2. Squalene has historically been harvested from shark liver oil, which is undesirable for a variety of reasons. In this study, we have demonstrated the use of a Synthetic Biology (yeast) production platform to generate squalene and novel triterpene oils, all of which are equally as efficacious as vaccine adjuvants based on physiochemical properties and immunomodulating activities in a mouse model. These Synthetic Biology adjuvants also elicited similar IgG1, IgG2a, and total IgG levels compared to marine and commercial controls when formulated with common quadrivalent influenza antigens. Injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.

Improving the baculovirus expression vector system with vankyrin-enhanced technology.

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex-type N-glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P-vank-1), which encodes an anti-apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High FiveTM cells, and SfSWT-4 cells, which can produce glycoproteins with complex-type N-glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N-glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P-vank-1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT-4 cells were infected with a vankyrin-encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin-expressing cells were combined with a vankyrin-encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin-encoding baculovirus vectors. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1496-1507, 2017.

Nonsurgical embryo transfer device compared with surgery for embryo transfer in mice.

The use of a murine nonsurgical embryo transfer (NSET) device had been described previously for the transfer of blastocysts, morulae, DNA-microinjected embryos, and embryonic stem cell-containing embryos to create genetically modified mice. However, physiologic effects of the NSET device and traditional surgical methods had not been compared directly. Here we used electrocardiography and fecal corticosterone levels to monitor pseudopregnant mice that underwent anesthesia only, the NSET procedure with or without anesthesia, or surgery. These procedures were performed without the use of actual embryos, to focus on effects of the procedures themselves rather than on any physiologic effects due to the deposition of embryos. As compared with surgery and anesthesia, the NSET procedure was associated with less fluctuation in cardiac rhythm and lower levels of the stress biomarker fecal corticosterone. These results indicate that use of the NSET device avoids these physi- ological perturbations as well as other disadvantages of surgery (for example, postoperative pain and need for postoperative analgesia) and therefore provides a valuable refinement of existing mouse embryo transfer procedures.