• Journal of Embryo Transfer
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• v.33 no.4
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• pp.245-254
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• 2018

Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of ${\alpha}$-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

• Food Science of Animal Resources
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• v.40 no.5
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• pp.852-859
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• 2020

The muscle stem cells of domestic animals are of interest to researchers in the food and biotechnology industries for the production of cultured meat. For producing cultured meat, it is crucial for muscle stem cells to be efficiently isolated and stably maintained in vitro on a large scale. In the present study, we aimed to optimize the method for the enrichment of pig muscle stem cells using a magnetic-activated cell sorting (MACS) system. Pig muscle stem cells were collected from the biceps femoris muscles of 14 d-old pigs of three breeds [Landrace×Yorkshire×Duroc (LYD), Berkshire, and Korean native pigs] and cultured in skeletal muscle growth medium-2 (SkGM-2) supplemented with epidermal growth factor (EGF), dexamethasone, and a p38 inhibitor (SB203580). Approximately 30% of total cultured cells were nonmyogenic cells in the absence of purification in our system, as determined by immunostaining for cluster of differentiation 56 (CD56) and CD29, which are known markers of muscle stem cells. Interestingly, following MACS isolation using the CD29 antibody, the proportion of CD56⁺/CD29⁺ muscle stem cells was significantly increased (91.5±2.40%), and the proportion of CD56 single-positive nonmyogenic cells was dramatically decreased. Furthermore, we verified that this method worked well for purifying muscle stem cells in the three pig breeds. Accordingly, we found that CD29 is a valuable candidate among the various marker genes for the isolation of pig muscle stem cells and developed a simple sorting method based on a single antibody to this protein.