Principle of Genetic Engineering

Nama : Arya Wira Wardhana

NPM : 21025010035

Class : A

Principle of Genetic Engineering

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:  how common elements of current technologies include the need for a chromosome break to occur, the use of specific and sensitive genotyping assays to detect altered genomes, and 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

There are many types of genetic modifications that can be made to the genome. The ability to specifically target locations in the genome has expanded our ability to make changes that include knockouts (DNA sequence deletions), knockins (DNA sequence insertions), and replacements (replacement of DNA sequences with exogenous sequences). Deletions in the genome can be used to knockout gene expression  Short deletions in the genome can be used to remove tein structure/function by changing coding sequences . Insertion of new genomic information can be used to knock in a variety of genetic elements. Knockins are also powerful approaches for modifying genes. Just as genomic deletions can be used to change gene function, knockins can be used to block gene function by inserting fluorescent reporter genes such as eGFP or mCherry, in such a way as to knock out the gene at the insertion  It is also possible to knock in fluorescent protein reporter genes, without knocking out the targeted gene Just as fluorescent proteins can be used to label proteins and cells, short knockins of epitope tags in proteins can be used to label proteins for detection with antibodies . Replacement of DNA sequences in the genome can be used to achieve two purposes at the same time, such as blocking gene function, while activating the function of a new gene such as the lacZ reporter Large-scale sequence replacements are possible with mouse ES cell technology, such as the replacement of the mouse immunoglobulin locus with the human immunoglobulin locus to produce a “humanized” mouse Furthermore, very small replacements of single nucleotides can be used to model point mutations that are suspected of causing human disease . A special type of DNA sequence replacement is the conditional allele. Conditional alleles permit normal gene expression until the site-specific Cre recombinase removes a loxP-flanked critical exon to produce a “floxed” (flanked by loxP) exon. Cre recombinase recognizes 34 bp loxP (locus of recombination) elements, and catalyzes recombination between the two loxP sites Therefore, deletion of the critical exon causes a premature termination codon to occur in the mRNA transcript, triggering its nonsense-mediated decay and failure to make a protein Engineering conditional alleles was the approach used by the international knockout mouse project Mice with cell- and tissue-specific Cre recombinase expression are an important resource for the research community  Other site-specific recombinases, such as FLP, Dre, and Vika, that work on the same principle have also been applied to mouse models  Recombinase knockins can be designed to knock out the endogenous gene or preserve its function  A variation in the conditional allele is the inducible allele, which is silent until its expression is activated by Cre recombinase . For example, reporter models can activate the expression of a fluorescent protein , change fluorescent reporter protein colors from red to green, or use a combinatorial approach to produce up to 90 fluorescent colors  Another type of inducible allele is the FLEX allele. FLEX genes are Cre-dependent gene switches based on the use of heterotypic loxP sites. In one application that combined Cre and FLP recombinases, it was demonstrated that a gene inactivated in ES cells by a gene trap could be switched back on and then switched offagain. In another application of heterotypic loxP sites in mouse ES cells, it was demonstrated that genes could be made conditional by inversion (COIN). This application has been used to produce mice with conditional genes for point mutations  and has been applied to produce conditional single exon genes that lack critical exons by definition