Direct injection of CRISPR/Cas9 into zygotes enables the production of genetically modified nonhuman primates (NHPs) essential for modeling specific human diseases, such as Usher syndrome, and for developing novel therapeutic strategies. Usher syndrome is a rare genetic disease that causes loss of hearing, retinal degeneration, and problems with balance, and is attributed to a mutation in MYO7A, a gene that encodes an uncommon myosin motor protein expressed in the inner ear and retinal photoreceptors. To produce an Usher syndrome type 1B (USH1B) rhesus macaque model, we disrupted the MYO7A gene in developing zygotes. Identification of appropriately edited MYO7A embryos for knockout embryo transfer requires sequence analysis of material recovered from a trophectoderm (TE) cell biopsy. However, the TE biopsy procedure is labor intensive and could adversely impact embryo development. Recent studies have reported using cell-free DNA (cfDNA) from embryo culture media to detect aneuploid embryos in human in vitro fertilization (IVF) clinics. The cfDNA is released from the embryo during cell division or cell death, suggesting that cfDNA may be a viable resource for sequence analysis. Moreover, cfDNA collection is not invasive to the embryo and does not require special tools or expertise. We hypothesized that selection of appropriate edited embryos could be performed by analyzing cfDNA for MYO7A editing in embryo culture medium, and that this method would be advantageous for the subsequent generation of genetically modified NHPs. The purpose of this experiment is to determine whether cfDNA can be used to identify the target gene mutation of CRISPR/Cas9 injected embryos. In this study, we were able to obtain and utilize cfDNA to confirm the mutagenesis of MYO7A, but the method will require further optimization to obtain better accuracy before it can replace the TE biopsy approach.

The optical wide-field patrol network (OWL-Net) is a Korean optical surveillance system that tracks and monitors domestic satellites. In this study, a batch least squares algorithm was developed for optical measurements and verified by Monte Carlo simulation and covariance analysis. Potential error sources of OWL-Net, such as noise, bias, and clock errors, were analyzed. There is a linear relation between the estimation accuracy and the noise level, and the accuracy significantly depends on the declination bias. In addition, the time-tagging error significantly degrades the observation accuracy, while the time-synchronization offset corresponds to the orbital motion. The Cartesian state vector and measurement bias were determined using the OWL-Net tracking data of the KOMPSAT-1 and Cryosat-2 satellites. The comparison with known orbital information based on two-line elements (TLE) and the consolidated prediction format (CPF) shows that the orbit determination accuracy is similar to that of TLE. Furthermore, the precision and accuracy of OWL-Net observation data were determined to be tens of arcsec and sub-degree level, respectively.

Estrogen related receptor β(Esrrb)는 오르판 수용체 중 하나로 전분화능 관련유전자인 Oct4와 Nanog의 발현을 조절함으로써 줄기세포의 미분화를 유지시키고, 지속적인 자기 복제를 가능케 하는 유전자로 알려져 있다. 또한 Feng 등 (2009)은 체세포에 Oct4, Sox2와 함께 Esrrb 유전자를 함께 도입하면, 유전자가 변형된 체세포가 배아 줄기세포와 유사한 유도만능줄기세포로 리프로그래밍(reprograming)되어 진다는 결과를 보고한 바 있다. 본 연구에서는 인간 ESRRB 단백질을 양수유래줄기세포 내로 직접도입하는 방법을 개발하고, 이를 통해 전분화능 관련유전자의 기능 조절을 확인하고자 하였다. 클로닝 된 인간 short-form ESRRB를 세포투과 펩타이드(cell-penetrating peptide, CPP)의 일종인 R7(아르기닌 7개)에 접합(Fusion)하였고, 합성단백질 (R7-ESRRB-His6)의 형태로 배양중인 인간 양수 유래 줄기세포에 처리하여 세포내로 도입하였다. R7-ESRRB-His6 단백질은 5시간 내에 세포막을 통과하였고, 24시간 내에 핵 내로 이동하였다. 또한 핵 내로 이동한 ESRRB 단백질은 OCT4와 NANOG 유전자의 발현을 증가시켰을 뿐만 아니라, 또 다른 전분화능 관련유전자인 SOX2의 발현도 함께 증가시킨다는 것을 확인하였다. 이상의 결과는 세포투과 펩타이드와 유전자의 접합을 통해 생산된 R7-ESRRB-His6 합성단백질이 양수유래줄기세포내로 원활하게 도입되는 것을 확인하였고, 유전자의 변형 없이 전분화능 관련유전자의 기능을 조절할 수 있는 방법임을 확인하였다.