Highly Efficient Knockin in Human iPS Cells and Rat Embryos by CRISPR/Cas9 Molecular Optimization

The CRISPR/Cas9 system is now the gold standard for the generation of genetically modified cell and animal models but knockin is a bottleneck. One reason could be that there is no consensus regarding the concentrations of its components to be used. Here, we defined optimal Cas9 protein, guide RNA and short donor DNA concentrations on a GFP to BFP conversion model of human induced pluripotent stem cells and point mutations on rat transgenic embryos. With a molecular rational approach of the CRISPR/Cas9 system and study of ribonucleoprotein complex formation by nanodifferential scanning fluorimetry, we defined that Cas9/guide RNA 1/1 molar ratio with 0.2µM and 0.4µM of Cas9, coupled with 2µM of ssODN are sufficient for optimal and high knockin frequencies in rat embryos and human induced pluripotent stem cells, respectively. These optimal conditions use lower concentrations of CRISPR reagents to form the RNP complex than most conditions published while achieving 50% of knockin. This study allowed us to reduce costs and toxicity while improving editing and knockin efficacy on two particularly key models to mimic human diseases.

Toxic Effect of Khat in Rat Embryos and Fetuses

Khat (Catha edulis Forsk) is a plant consumed by many people in Eastern Africa, including Ethiopia, and Southern Arabia to be stimulated. There are several human and animal studies on khat that provide information about its toxic effects. However, the potential toxic effects of khat on embryos and fetuses have not been elucidated. The aim of the present study was to investigate the embryotoxic and fetotoxic effects of khat exposure during the earliest period of gestation in rats. Pregnant Wistar albino rats were treated with khat extract at 250, 500, and 750 mg/kg doses from day 6 through day 12 of gestation. The treatment was delivered by gavage. Embryos and fetuses were recovered on gestational day 12 or day 20, respectively, and were quantitatively and qualitatively assessed for developmental anomalies. Placentae from the treatment and control groups were investigated for histopathological effects. Results of the present study showed that khat exposure during pregnancy had dose-dependent toxic effects in rat embryos and fetuses. Prenatal growth retardation such as reduced fetal weight and crown-rump length was observed in near-term fetuses, especially, in animals treated with the highest dose of khat ( p < 0.05 ). Growth retardation and developmental anomalies were also observed in day 12 embryos of khat-treated rats. Maternal weight gain of the khat-treated group was also significantly lower than the control group. Cytolysis, decidual hypoplasia, and atrophy were observed in the placenta of the khat-treated rats. Findings of the present study revealed, for the first time, that exposure of pregnant rat to crude extract of khat causes embryotoxic and fetotoxic effects.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol, 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2,613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18,467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

Teratogenic Effect of High Dose of Syzygium guineense (Myrtaceae) Leaves on Wistar Albino Rat Embryos and Fetuses

Syzygium guineense is an important medicinal plant effective against hypertension, diabetes mellitus, and cancer but with no evidence of its teratogenicity. This study was planned to investigate the teratogenic potential of S. guineense leaves on rat embryos and fetuses. Five groups of Wistar albino rats, each consisting of ten pregnant rats, were used as experimental animals. Groups I-III rats were treated with 250, 500, and 1000 mg/kg of hydroethanolic extract of S. guineense leaves, and groups IV and V were control and ad libitum control, respectively. Rats were treated during day 6–12 of gestation. Embryos and fetuses were retrieved at day 12 and day 20 of gestation, respectively. The embryos were assessed for developmental delays and growth retardation. The fetuses were examined for gross external, skeletal, and visceral anomalies. In 12-day old rat embryos, crown-rump length, number of somites, and morphological scores were significantly reduced by the treatment of 1000 mg/kg of the extract. The external morphological and visceral examinations of rat fetuses did not reveal any detectable structural malformations in the cranial, nasal, oral cavities, and visceral organs. The ossification centers of fetal skull, vertebrae, hyoid, forelimb, and hindlimb bones were not significantly varied across all groups. However, even if not statistically significant, high-dose treated rat fetuses had a reduced number of ossification centers in the sternum, caudal vertebrae, metatarsal, metacarpal, and phalanges. Treatment with the hydroethanolic extract of S. guineense leaves produced no significant skeletal and soft tissue malformations. The plant extract did not produce significant teratogenic effects on rat embryos/fetuses up to 500 mg/kg doses but retarded the growth of embryos at high dose (1000 mg/kg) as evidenced by decreased crown-rump length, number of somites, and morphological scores. Therefore, it is not advisable to take large doses of the plant during pregnancy.

Midgut development in rat embryos using microcomputed tomography

The development of the mammalian gut was first described more than a century ago. Since then, it has been believed that a series of highly orchestrated developmental processes occur before the intestine achieves its final formation. The key steps include the formation of the umbilicus, the so-called “physiological herniation” of the midgut into the umbilical cord, an intestinal “rotation”, and the “return of the gut” into the abdominal cavity. However, this sequence of events is predominantly based on histological sections of dissected embryos, a 2D technique with methodological limitations. For a better understanding of spatial relationships in the embryo, we utilized microcomputed tomography (µCT), a nondestructive 3D imaging method. Here, we show the detailed processes and mechanisms of intestinal development in rat embryos, including the development of the umbilicus, the formation of loops inside the umbilical coelom, and the subsequent shift of these loops into the abdominal cavity. Our 3D datasets of developing intestines will substantially advance the understanding of normal mammalian midgut embryology and offer new possibilities to reveal unknown mechanisms in the pathogenesis of congenital disorders.

Toxicity of Methanolic Extracts of Seeds of Moringa stenopetala, Moringaceae in Rat Embryos and Fetuses

Moringa stenopetala is a medicinal plant that has been used in Ethiopian traditional medicine as a remedy for the treatment of hypertension, diabetes, and stomach pain. The study is aimed at assessing the toxicity of the methanol extracts of the seeds of Moringa stenopetala on the developing embryo and fetuses of rats. The seeds of Moringa were extracted by maceration using 80% methanol. The extract (250–1000 mg/kg) was orally administered to pregnant Swiss albino rats from days 6 to12 of gestation. Embryos and fetuses were recovered by laparotomy on gestational day 12 and day 20, respectively, and were assessed for developmental anomalies. On day 20, significant prenatal growth retardation such as reduced litter weight and crown-rump length were observed in near term fetuses of 1000 mg/kg treated rats. Litter weight in 1000 mg/kg and pair-fed control groups was 2.41 g ± 0.108 and 3.08 g ± 0.093 , respectively. Delay in the development of an otic, optic, and olfactory system, as well as a reduction in a number of branchial bars, occurred on day 12 embryos of 1000 mg/kg treated rats. The rate of fetal resorption in 1000 mg/kg and pair-fed control groups was 1.6 ± 0.55 and 0.42 ± 0.52 , respectively. There was also a high incidence of fetal death in the 1000 mg/kg treated group but it was not statistically significant. The offspring’s of Moringa-treated rats did not show gross external malformations at all doses. These findings suggest that the methanol seed extract of Moringa stenopetala is not safe to rat embryos and fetuses. Its toxic effects were evidenced by a significant delay in embryonic and fetal development and an increase in fetal resorptions and fetal death.