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Principles of Genetic Engineering

by 1,2,†, 1,3,† and 4,5,*
1
Biomedical Research Core Facilities, Vector Core, University of Michigan, Ann Arbor, MI 48109, USA
2
Department of Internal Medicine, Division of Rheumatology, University of Michigan, Ann Arbor, MI 48109, USA
3
Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA
4
Biomedical Research Core Facilities, Transgenic Animal Model Core, University of Michigan, Ann Arbor, MI 48109, USA
5
Department of Internal Medicine, Division of Genetic Medicine, University of Michigan, Ann Arbor, MI 48109, USA
*
Author to whom correspondence should be addressed.
These authors contributed to the work equally.
Genes 2020, 11(3), 291; https://doi.org/10.3390/genes11030291
Received: 31 December 2019 / Revised: 28 February 2020 / Accepted: 6 March 2020 / Published: 10 March 2020
(This article belongs to the Special Issue Prospects in Transgenic Technology 2020)
Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields. View Full-Text
Keywords: CRISPR/Cas9; embryonic stem (ES) cells; genetic engineering; gene targeting; homologous recombination; microinjection; retroviruses; transgenic mice; transgenic rats; transposons; vectors CRISPR/Cas9; embryonic stem (ES) cells; genetic engineering; gene targeting; homologous recombination; microinjection; retroviruses; transgenic mice; transgenic rats; transposons; vectors
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MDPI and ACS Style

Lanigan, T.M.; Kopera, H.C.; Saunders, T.L. Principles of Genetic Engineering. Genes 2020, 11, 291. https://doi.org/10.3390/genes11030291

AMA Style

Lanigan TM, Kopera HC, Saunders TL. Principles of Genetic Engineering. Genes. 2020; 11(3):291. https://doi.org/10.3390/genes11030291

Chicago/Turabian Style

Lanigan, Thomas M.; Kopera, Huira C.; Saunders, Thomas L. 2020. "Principles of Genetic Engineering" Genes 11, no. 3: 291. https://doi.org/10.3390/genes11030291

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