Gene delivery to fertilized eggs is often the first step in creation of transgenic animals, CRISPR knock-out, or early developmental studies. The zona pellucida, a hardened glycoprotein matrix surrounding the mammalian fertilized eggs, often complicates gene delivery by forming a barrier against transfection reagents and viruses. High efficiency techniques to perforate or penetrate the zona allow for access and gene delivery to fertilized eggs. However, these techniques often rely on highly skilled technologists, are costly, and require specialized equipment for micromanipulation, laser perforation, or electroporation. Here, we report that adenoassociated viruses (AAVs) with serotypes 1 or DJ can efficiently diffuse across the zona to deliver genes without any manipulations to fertilized eggs. We observe lowered rates of embryo development after treatment of embryos with all AAV serotypes. However, we were able to reduce adverse effects on embryo development by exposing embryos to AAVs at later stages of in vitro development. AAVs have low immune response and do not incorporate into their host chromosomes to cause insertional mutations. Hence, AAVs can serve as a highly effective tool for transient delivery of genes to fertilized mammalian eggs.

Background: Hyperoxia or clinical oxygen (O-2) therapy is known to result in increased oxidative burden. Therefore, understanding susceptibility to hyperoxia exposure is clinically important. Bone morphogenetic proteins (BMPs) 2 and 4 are involved in cardiac development and may influence responses to hyperoxia.Methods: Bmp2(+/-), Bmp4(+/-) and wild-type mice were exposed to hyperoxia (100% O-2) for 24 hrs. Electrocardiograms (ECG) were recorded before and during exposure by radio-telemetry.Results: At baseline, a significantly higher low frequency (LF) and total power (TP) heart rate variability (HRV) were found in Bmp2(+/-) mice only (p<0.05). Twenty-four hours hyperoxia-induced strain-independent reductions in heart rate, QTcB and ST-interval and increases in QRS, LF HRV and standard deviation of RR-intervals were observed. In Bmp4(+/-) mice only, increased PR-interval (PR-I) (24 hrs), P-wave duration (P-d; 18 and 21-24 hrs), PR-I minus P-d (PR-Pd; 24 hrs) and root of the mean squared differences of successive RR-intervals (24 hrs) were found during hyperoxia (p50.05).Discussion: Elevated baseline LF and TP HRV in Bmp2(+/-) mice suggests an altered autonomic nervous system regulation of cardiac function in these mice. However, this was not related to strain specific differences in responses to 24 hrs hyperoxia. During hyperoxia, Bmp4(+/-) mice were the most susceptible in terms of atrioventricular conduction changes and risk of atrial fibrillation, which may have important implications for patients treated with O-2 who also harbor Bmp4 mutations. This study demonstrates significant ECG and HRV responses to 24 hrs hyperoxia in mice, which highlights the need to further work on the genetic mechanisms associated with cardiac susceptibility to hyperoxia.

BackgroundProlonged hyperoxia (96hrs) leads to adverse murine cardiac responses, but clinical therapy with high O2 levels is often short‐term. Therefore, understanding susceptibility to short‐term hyperoxia exposure is clinically important. Bone morphogenetic proteins (BMP) 2 and 4 are important in cardiac development and may influence cardiac responses to hyperoxia. We investigated cardiac responses to 24hrs hyperoxia in Bmp2+/− and Bmp4+/− mice.MethodsBmp2+/−, Bmp4+/− and WT littermates were exposed to 24hrs hyperoxia (100% O2). ECG was recorded before and during exposure.ResultsNo differences in baseline HR, QTcB, QRS, ST‐I, PR‐I, P‐wave duration (P‐d) or PR‐I/P‐d were found (P >;0.05). After ~18hrs hyperoxia, significant decreases in HR and increases in PR‐I and P‐d were found in Bmp4+/− mice only. Also, there were reductions in QTcB in the WT, but not in Bmp2+/− or Bmp4+/− mice after 17hrs hyperoxia (P <0.05).DiscussionResults suggest that developmental effects of Bmp2 or Bmp4 disruption did not affect basal cardiac function or were not detectable by ECG. During hyperoxia, Bmp4+/− mice were susceptible in terms of HR and atrioventricular conduction responses, which may have important implications for patients treated with O2 who also harbor Bmp4 mutations. Conversely, QTcB in Bmp2+/− or Bmp4+/− mice was not affected by hyperoxia, unlike WT mice, suggesting a protective effect in these genotypes.