Nokaut miševi: klasični vs. moderni pristup

undergraduate thesis

• Title (english): Knockout mice: classic vs. modern approach
• Author: Roko Gvozdenica Šipić
• Mentor: Nenad Malenica (mentor)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-09-22, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

KO miševi jedan su od najvažnijih modelnih organizama za genetička istraživanja. Njihovim proučavanjem došlo se i dolazi do brojnih otkrića u pogledu funkcije gena, patologije bolesti, mehanizama genske regulacije i sličnog. Za neka otkrića koja su omogućila proizvodnju takvih organizama dodijeljena je i Nobelova nagrada. U ovom radu predstavljene su osnovne metode njihove proizvodnje uz pregled njihove efikasnosti. Klasične metode kreiranja knockout miševa nazivaju se gene trapping i gene targeting. Gene trapping sastoji se od nasumične ugradnje vektora s reporter genom u odabrani lokus genoma embrionalne matične stanice u kulturi s ciljem inaktivacije gena u tom lokusu. Tako transformirane matične stanice unose se u blastocistu i rezultiraju kimernim mišem, od kojega se povratnim križanjem dobiva KO miš. Gene targeting prva je metoda koja je omogućila ciljanu inaktivaciju gena. Također se provodi na embrionalnim matičnim stanicama u kulturi, a ugradnja se zasniva na homolognoj rekombinaciji između unešenog genetičkog materijala i lokusa od interesa. Najnovije metode transformiraju zigotu, što eliminira korak s kimernim mišem i povećava efikasnost proizvodnje, te skraćuje vrijeme koje je za nju potrebno. Te metode zasnivaju se na korištenju sustava ZFN, TALEN i CRISPR/Cas9. ZFN i TALEN dizajnirane su endonukleaze koje specifično prepoznaju genske lokuse pomoću DNA vezujućih domena i uvode dvolančane lomove endonukleazom FokI. CRISPR/Cas9 koristi bakterijski antivirusni kompleks Cas9 navođen sgRNA molekulom koji također uvodi dvolančani lom. Najnovija varijanta naziva prime editing uključuje korištenje Cas9 proteina s nikaznom aktivnošću i gotovo potpuno eliminira nespecifične reakcije. Sustav CRISPR/Cas9 nudi do tada neviđen stupanj efikasnosti u uvođenju KO mutacija uz razmjerno jednostavan i jeftin postupak.

Abstract (english)

KO mice are one of the most important model organisms for genetic research. Their study has lead to numerous discoveries regarding gene function, pathology of diseases, mechanisms of gene regulation and the like. For some discoveries that made it possible to produce such organisms, even the Nobel Prize was awarded. This paper presents the basic methods of their production with an overview of their efficiency. Classic methods of creating knockout mice are called gene trapping and gene targeting. Gene trapping consists of randomly embedding vectors with the reporter genome in the selected locus genome of embryonic stem cells in culture to inactivate genes in this locus. Thus, transformed stem cells are introduced into the blastocyst and result in a chimeric mouse, from which a KO mouse is obtained by reverse crossing. Gene targeting is the first method to enable targeted gene inactivation. It is also performed on embryonic stem cells in culture, and modification is based on homologous recombination between the entered genetic material and the locus of interest. The latest methods transform the zygote, which eliminates the step with the chimeric mouse and increases the efficiency of production, shortening the time required. These methods are based on the use of ZFN, TALEN and CRISPR/Cas9 systems. ZFN and TALEN are artificial endonucleases that specifically recognize gene loci using DNA binding domains and introduce double-stranded breaks using endonuclease FokI. CRISPR/Cas9 uses the bacterial antiviral complex Cas9 guided by a sgRNA molecule that also introduces double-strand breaks. The latest method is named prime editing and involves the use of Cas9 proteins with nickase activity, almost completely eliminating nonspecific reactions. The CRISPR/Cas9 system offers an unprecedented degree of efficiency in introducing KO mutations with a relatively simple and inexpensive process.

• Keywords: KO miševi; gene trapping; gene targeting; ZFN; TALEN; CRISPR/Cas9
• Keywords (english): KO mice; gene trapping; gene targeting; ZFN; TALEN; CRISPR/Cas9
• Language: croatian
• URN:NBN: urn:nbn:hr:217:597621
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: undergraduate; Academic / professional title: sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) molekularne biologije (sveučilišni/a prvostupnik/prvostupnica (baccalaureus/baccalaurea) molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-09-27 12:17:51