Abstract
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.
Highlights
Since the identification of DNA as the unit of heredity and the basis for the central dogma of molecular biology [1] that DNA makes RNA and RNA makes proteins, scientists have pursued experiments and methods to understand how DNA controls heredity
Compared with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) are easier to construct with publicly available resources [40,41], and TALENs have been adopted for use in mouse
Compared with ZFNs, TALENs are easier to construct with publicly available resources [40,41], and TALENs have been adopted for use in mouse and rat genome engineering in several laboratories and have rat genome engineering several laboratories that have produced knockout and knockin animal that produced knockoutinand knockin animal models
Summary
Since the identification of DNA as the unit of heredity and the basis for the central dogma of molecular biology [1] that DNA makes RNA and RNA makes proteins, scientists have pursued experiments and methods to understand how DNA controls heredity. Thousands of mouse models have resulted from that effort and have been used to better understand gene function and the bases of human genetic diseases [14] This project required high-throughput pipelines for the construction of vectors, including bacterial artificial chromosome (BAC) recombineering technology [13,15,16,17]. When the FokI endonuclease domains are brought together, a double-stranded DNA break occurs In this way, a TALEN heterodimer can be used to cause a sequence-specific chromosome break. Compared with ZFNs, TALENs are easier to construct with publicly available resources [40,41], and TALENs have been adopted for use in mouse and rat genome engineering in several laboratories and have rat genome engineering several laboratories that have produced knockout and knockin animal that produced knockoutinand knockin animal models [42,43,44,45,46].
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