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.

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.

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.

Effect of Campoletis sonorensis ichnovirus cys-motif proteins on Heliothis virescens larval development.

Polydnaviruses are obligate symbionts of some parasitic hymenopteran wasps responsible for modifying the physiology of their host lepidopteran larvae to benefit the endoparasite. Injection of Campoletis sonorensis ichnovirus (CsIV) into Heliothis virescens larvae alters larval growth, development and immunity but genes responsible for alterations of host physiology are not well described. Recent studies of polydnavirus genomes establish that these genomes encode families of related genes expressed in parasitized larvae. Here we evaluate five members of the CsIV cys-motif gene family for their ability to inhibit growth and development of lepidopteran larvae. To study the function of cys-motif proteins, recombinant proteins were produced from baculovirus expression vectors and injected or fed to H. virescens larvae in diet. rVHv1.1 was identified as the most potent protein tested causing a significant reduction in growth of H. virescens and Spodoptera exigua larvae. H. virescens larvae ingesting this protein also exhibited delayed development, reductions in pupation and increased mortality. Increased mortality was associated with chronic sub-lethal baculovirus infections. Taken together, these data indicate that the cys-motif proteins have pleiotropic effects on lepidopteran physiology affecting both development and immunity.

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.

Polydnavirus genes that enhance the baculovirus expression vector system.

The baculovirus expression vector system (BEVS) is a powerful and versatile system for protein expression, which has many advantages. However, a limitation of any lytic viral expression system, including BEVS, is that death and lysis of infected insect cells terminates protein production. This results in interruption of protein production and higher production costs due to the need to set up new infections, maintain uninfected cells, and produce pure viral stocks. Genetic methods to slow or prevent cell death while maintaining high-level, virus-driven protein production could dramatically increase protein yields. Several approaches have been used to improve the BEVS and increase the synthesis of functional proteins. Successful enhancement of the BEVS was obtained when various gene elements were added to the virus, secretion and posttranslational processing were modified, or protein integrity was improved. A gene family from the insect virus Campoletis sonorensis ichnovirus (CsIV) was discovered that delays lysis of baculovirus-infected cells, thereby significantly enhancing recombinant protein production in the BEVS system. By using the CsIV vankyrin gene family, protein production in the vankyrin-enhanced BEVS (VE-BEVS) was increased by a factor of 4- to 15-fold by either coexpressing the vankyrin protein from a dual BEVS or by providing its activity in trans by expressing the vankyrin protein from a stably transformed cell line. In sum, VE-BEVS is an enhancement of the existing BEVS technology that markedly improves protein expression levels while reducing the cost of labor and materials.

Potential uses of Cys-motif and other polydnavirus genes in biotechnology.

Exploiting the ability of insect pathogens, parasites, and predators to control natural and damaging insect populations is a cornerstone of biological control. Here we focus on an unusual group of viruses, the polydnaviruses (PDV), which are obligate symbionts of some hymenopteran insect parasitoids. PDVs have a variety of important pathogenic effects on their parasitized hosts. The genes controlling some of these pathogenic effects, such as inhibition of host development, induction of precocious metamorphosis, slowed or reduced feeding, and immune suppression, may have use for biotechnological applications. In this chapter, we consider the physiological functions of both wasp and viral genes with emphasis on the Cys-motif gene family and their potential use for insect pest control.