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CRISPR/Cas9 Electroporation in Sheep Zygotes with Laser Zona Drilling.

Record Type:	Electronic resources : Monograph/item
Title/Author:	CRISPR/Cas9 Electroporation in Sheep Zygotes with Laser Zona Drilling./
Author:	Cruz, Michelle Beatrice.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2018,
Description:	52 p.
Notes:	Source: Masters Abstracts International, Volume: 81-03.
Contained By:	Masters Abstracts International81-03.
Subject:	Animal sciences. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13424180
ISBN:	9781085736732

CRISPR/Cas9 Electroporation in Sheep Zygotes with Laser Zona Drilling.
Cruz, Michelle Beatrice.

CRISPR/Cas9 Electroporation in Sheep Zygotes with Laser Zona Drilling. - Ann Arbor : ProQuest Dissertations & Theses, 2018 - 52 p.

Source: Masters Abstracts International, Volume: 81-03.

Thesis (M.S.)--University of California, Davis, 2018.

This item is not available from ProQuest Dissertations & Theses.

CRISPR/Cas9 technology has revolutionized the gene editing world. CRISPR/Cas9 gene editing can be used in yeast, plants, and animals. Due to its high efficiency, CRISPR/Cas9 can be used to generate genome edited livestock. The current primary method for CRISPR/Cas9 delivery in zygotes is through microinjection, however, this practice is inefficient. Microinjection requires an experienced individual to inject a single zygote at a time, making it difficult to process a large group in a small period of time. While microinjection has proven to be a functional method for CRISPR/Cas9 delivery, there are other alternatives that may be more efficient and just as effective. In this study, CRISPR/Cas9 electroporation was optimized for use in sheep zygotes using the BIORAD Gene Pulser X-Cell as an alternative to microinjection. Using parthenogenically activated and in vitro fertilized zygotes we were able to optimize electroporation parameters in sheep zygotes to produce blastocysts containing targeted mutations; demonstrating that electroporation is a viable alternative to microinjection. Bi-allelic and monoallelic mutations in the PDX1 gene were produced in sheep embryos through the use of laser zona drilling (LZD) to thin the zona pellucida (ZP). In addition, we were able to obtain a higher percentage of mutation efficiency in comparison to intact zona (IZ) zygotes. In the LZD mutated blastocysts, there was also a higher percent of bi-allelic deletions than in the IZ treatment. CRISPR/Cas9 electroporation is an efficient and simple delivery method to produce deletions or insertions in sheep. This method can be applied to other livestock species. The use of CRISPR/Cas9 electroporation can be useful in various applications to produce gene edited animals on a larger scale.

ISBN: 9781085736732Subjects--Topical Terms:

3174829
Animal sciences.
Subjects--Index Terms:

CRISPR/Cas9

CRISPR/Cas9 Electroporation in Sheep Zygotes with Laser Zona Drilling.
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300 $a 52 p.
500 $a Source: Masters Abstracts International, Volume: 81-03.
500 $a Advisor: Ross, Pablo J.
502 $a Thesis (M.S.)--University of California, Davis, 2018.
506 $a This item is not available from ProQuest Dissertations & Theses.
506 $a This item must not be sold to any third party vendors.
520 $a CRISPR/Cas9 technology has revolutionized the gene editing world. CRISPR/Cas9 gene editing can be used in yeast, plants, and animals. Due to its high efficiency, CRISPR/Cas9 can be used to generate genome edited livestock. The current primary method for CRISPR/Cas9 delivery in zygotes is through microinjection, however, this practice is inefficient. Microinjection requires an experienced individual to inject a single zygote at a time, making it difficult to process a large group in a small period of time. While microinjection has proven to be a functional method for CRISPR/Cas9 delivery, there are other alternatives that may be more efficient and just as effective. In this study, CRISPR/Cas9 electroporation was optimized for use in sheep zygotes using the BIORAD Gene Pulser X-Cell as an alternative to microinjection. Using parthenogenically activated and in vitro fertilized zygotes we were able to optimize electroporation parameters in sheep zygotes to produce blastocysts containing targeted mutations; demonstrating that electroporation is a viable alternative to microinjection. Bi-allelic and monoallelic mutations in the PDX1 gene were produced in sheep embryos through the use of laser zona drilling (LZD) to thin the zona pellucida (ZP). In addition, we were able to obtain a higher percentage of mutation efficiency in comparison to intact zona (IZ) zygotes. In the LZD mutated blastocysts, there was also a higher percent of bi-allelic deletions than in the IZ treatment. CRISPR/Cas9 electroporation is an efficient and simple delivery method to produce deletions or insertions in sheep. This method can be applied to other livestock species. The use of CRISPR/Cas9 electroporation can be useful in various applications to produce gene edited animals on a larger scale.
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653 $a Electroporotation
653 $a Gene Editing
653 $a Laser zona drilling
653 $a Sheep
653 $a Zygotes
690 $a 0475
710 2 $a University of California, Davis. $b Animal Biology. $3 1669099
773 0 $t Masters Abstracts International $g 81-03.
790 $a 0029
791 $a M.S.
792 $a 2018
793 $a English
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