Efficient production of GGTA1 knockout porcine embryos using a modified handmade cloning (HMC) method

Ramesh Kumbha, Nora Hosny, Anders Matson, Magie Steinhoff, Bernhard J. Hering, Christopher Burlak

Research output: Contribution to journalArticle

Abstract

Handmade cloning is a zona-free nuclear transfer approach and an economical, efficient, and simple micromanipulation-free alternative to dolly based traditional cloning (TC). In this study, based on handmade cloning with minor modifications, an optimized bi-oocyte fusion (BOF) cloning method was established to produce GGTA1 KO porcine embryos using the CRISPR/Cas9 gene editing system. The GGTA1 gene is responsible for the generation of Gal epitopes on the surface of porcine cells, triggering hyperacute immune rejection in preclinical porcine-to-human xenotransplantation. The purpose of the present study is to establish an efficient protocol for activation of porcine oocyte cytoplast-fibroblast fused constructs developed to GGTA1 KO blastocysts by the zona-free bi-oocyte fusion cloning method. High percentages of cleavage (90 ± 2.6%) and blastocyst rates (39 ± 4.0%) were achieved upon treatment with demecolcine-assisted oocyte enucleation followed by 6 V alternating current for proper alignment and single-step fusion technique using a single direct current pulse of 1.0 kV/cm for 9 μs duration, compared to the double-step fusion method with combined chemical activation using thimerosal and dithiothreitol. Overall blastocyst rate was higher for oocyte enucleation by demecolcine (0.4 μg/ml) and 45 min incubation (42 ± 1.5%) compared to without demecolcine incubation followed by complete chemical thimerosal/dithiothreitol activation (33 ± 1.1%). The blastocyst rate (39 ± 1.0%) was found to be significantly higher 1 h post-electrofusion, compared to at 0 and 4 h (28 ± 1.5 and 6 ± 1.5%, respectively). Blastocyst development rates for GGTA1 knockout embryos (38 ± 1.76%) were comparable to those obtained with wild-type embryos (41.1 ± 0.67%). In conclusion, we achieved high overall efficiency in production of GGTA1 KO blastocysts by modified HMC protocol.

Original languageEnglish (US)
Pages (from-to)59-68
Number of pages10
JournalResearch in veterinary science
Volume128
DOIs
StatePublished - Feb 2020

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Blastocyst
blastocyst
Organism Cloning
molecular cloning
embryo (animal)
Swine
Embryonic Structures
Oocytes
Demecolcine
oocytes
swine
thimerosal
Thimerosal
dithiothreitol
Dithiothreitol
Herpes Zoster
Clustered Regularly Interspaced Short Palindromic Repeats
methodology
Micromanipulation
electrofusion

PubMed: MeSH publication types

  • Journal Article

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Efficient production of GGTA1 knockout porcine embryos using a modified handmade cloning (HMC) method. / Kumbha, Ramesh; Hosny, Nora; Matson, Anders; Steinhoff, Magie; Hering, Bernhard J.; Burlak, Christopher.

In: Research in veterinary science, Vol. 128, 02.2020, p. 59-68.

Research output: Contribution to journalArticle

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abstract = "Handmade cloning is a zona-free nuclear transfer approach and an economical, efficient, and simple micromanipulation-free alternative to dolly based traditional cloning (TC). In this study, based on handmade cloning with minor modifications, an optimized bi-oocyte fusion (BOF) cloning method was established to produce GGTA1 KO porcine embryos using the CRISPR/Cas9 gene editing system. The GGTA1 gene is responsible for the generation of Gal epitopes on the surface of porcine cells, triggering hyperacute immune rejection in preclinical porcine-to-human xenotransplantation. The purpose of the present study is to establish an efficient protocol for activation of porcine oocyte cytoplast-fibroblast fused constructs developed to GGTA1 KO blastocysts by the zona-free bi-oocyte fusion cloning method. High percentages of cleavage (90 ± 2.6{\%}) and blastocyst rates (39 ± 4.0{\%}) were achieved upon treatment with demecolcine-assisted oocyte enucleation followed by 6 V alternating current for proper alignment and single-step fusion technique using a single direct current pulse of 1.0 kV/cm for 9 μs duration, compared to the double-step fusion method with combined chemical activation using thimerosal and dithiothreitol. Overall blastocyst rate was higher for oocyte enucleation by demecolcine (0.4 μg/ml) and 45 min incubation (42 ± 1.5{\%}) compared to without demecolcine incubation followed by complete chemical thimerosal/dithiothreitol activation (33 ± 1.1{\%}). The blastocyst rate (39 ± 1.0{\%}) was found to be significantly higher 1 h post-electrofusion, compared to at 0 and 4 h (28 ± 1.5 and 6 ± 1.5{\%}, respectively). Blastocyst development rates for GGTA1 knockout embryos (38 ± 1.76{\%}) were comparable to those obtained with wild-type embryos (41.1 ± 0.67{\%}). In conclusion, we achieved high overall efficiency in production of GGTA1 KO blastocysts by modified HMC protocol.",
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