Development of PCR based approach to detect potential mosaicism in porcine embryos

Cho, Jongki; Uh, Kyungjun; Ryu, Jae-Yong; Fang, Xun; Bang, Seonggyu; Lee, Kiho

doi:10.12750/jarb.35.4.323

Cited by 5 publications

(4 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It may be difficult to determine mutations in the whole embryo since TE-biopsied samples only have 10-15 cells, less than 10% of the whole embryo cell population. Similar results were reported in other species, such as sheep and pigs (Vilarino et al, 2018;Cho et al, 2020).…”

Section: Resultssupporting

confidence: 90%

Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos

Ryu

Burch

Mishler

et al. 2022

J Anim Reprod Biotechnol

View full text Add to dashboard Cite

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.

show abstract

Section: Resultssupporting

confidence: 90%

Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos

Ryu

Burch

Mishler

et al. 2022

J Anim Reprod Biotechnol

View full text Add to dashboard Cite

show abstract

“…The relative levels of gene expression in different groups were analyzed; skin samples (including tissues regenerated during wound healing) were collected immediately after anesthesia and preserved at -80 °C until processing. The sample collection and evaluation of the relative level of gene expression were performed according to a previously described method [ 55 , 67 ]. A quantitative real-time polymerase chain reaction (qRT-PCR) kit (BioFACT, Daejeon, South Korea) was used according to the manufacturer's protocol.…”

Section: Methodsmentioning

confidence: 99%

RETRACTED: Centella asiatica extract in carboxymethyl cellulose at its optimal concentration improved wound healing in mice models

Tanga¹,

Bang²,

Fang³

et al. 2022

Heliyon

Self Cite

View full text Add to dashboard Cite

“…Polymerase chain reaction (PCR)-based techniques are best due to their more quantitative nature, but their major limitation is the need for prior knowledge of the gene sequences to be assessed [ 25 , 200 ]. Most studies indicate that sperm RNA levels can be used to study the past events of spermatogenesis, sperm function, and successful fertilization in cattle and pig [ 198 , 201 ].…”

Section: Sperm Transcriptome Assay (Gene Expression)mentioning

confidence: 99%

Semen evaluation: methodological advancements in sperm quality-specific fertility assessment

et al. 2021

Self Cite

View full text Add to dashboard Cite

Assessment of male fertility is based on the evaluation of sperm. Semen evaluation measures various sperm quality parameters as fertility indicators. However, semen evaluation has limitations, and it requires the advancement and application of strict quality control methods to interpret the results. This article reviews the recent advances in evaluating various sperm-specific quality characteristics and methodologies, with the help of different assays to assess sperm-fertility status. Sperm evaluation methods that include conventional microscopic methods, computer-assisted sperm analyzers (CASA), and flow cytometric analysis, provide precise information related to sperm morphology and function. Moreover, profiling fertility-related biomarkers in sperm or seminal plasma can be helpful in predicting fertility. Identification of different sperm proteins and diagnosis of DNA damage has positively contributed to the existing pool of knowledge about sperm physiology and molecular anomalies associated with different infertility issues in males. Advances in methods and sperm-specific evaluation has subsequently resulted in a better understanding of sperm biology that has improved the diagnosis and clinical management of male factor infertility. Accurate sperm evaluation is of paramount importance in the application of artificial insemination and assisted reproductive technology. However, no single test can precisely determine fertility; the selection of an appropriate test or a set of tests and parameters is required to accurately determine the fertility of specific animal species. Therefore, a need to further calibrate the CASA and advance the gene expression tests is recommended for faster and field-level applications.

show abstract

Development of PCR based approach to detect potential mosaicism in porcine embryos

Cited by 5 publications

References 28 publications

Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos

Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos

RETRACTED: Centella asiatica extract in carboxymethyl cellulose at its optimal concentration improved wound healing in mice models

Semen evaluation: methodological advancements in sperm quality-specific fertility assessment

Contact Info

Product

Resources

About