Reverse genetics by gene knock down / knock out using morpholinos.
What are morpholinos and how do they function?
DNA bases attached to a backbone of methylenemorpholine rings -> linked through phosphodiamidate groups
A morpholino binds to its antisense mRNA and thereby blocks it (=RNAi)
What kinds of morpholinos are there?
ATG morpholinos -> bind to AUG start codon and thereby blocks every mRNA -> used to test the morpholinos blocking activity (with western blot analysis)
splice-site-morpholinos: block the mRNA processing and can be used to research alternative splicing or partly GoF or LoF variants
splice site donors —> exon-intron, insertion of intron
splice site acceptors —> intron-exon, deletion of exon
specific antisense morpholinos that target specific transcripts?
Transcriptional gene silencing and genome engineering with targetable Nucleases (FokI)
by ZFNs [Zink finger nucleases]
by TALENs [transcription-activator–like effector nucleases]
by CRISPRs [type II prokaryotic clustered regularly interspaced short palindromic repeats]
What is needed:
specific recognition of long target sequences (long enough for unique occurrence in a eukaryotic genome)
adaptability for retargeting to user-defined sequences.
FokI Nuclease: From Flavobacterium okeanokoites, is a bacterial restriction endonuclease consisting of an N-terminal DNAbinding domain and a non-specific DNA cleavage domain at the C-terminus. -> part of the DNA repair mechanism -> introducing double strand breaks
TALEN -> have a functional domain that binds the DNA the AA code can be altered so it is specific for different DNA bases (different AA determine specificity for Cytosin / Guanin / Thymin / Adenin) -> the TALEN binding is then combined with Fokl nuclease domain so the DNA is cut!
CRISPR / CAS system is a similar but more efficient method -> a bacterial small RNA-based defense system -> the viral RNA templates are safe the CRISPR locus “database” then combined with the CAS9 the antisense is detected and then cleaved at an exact location (20 bp)
-> RNA only expression method most commonly used = guideRNA + CAS9 protein
CAS9 can also be linked to repressor or activator, fluorecent or epicgenetic modifier domain for genetic regulation manipulation, visualizetion …
Proposed mechanism for mutationinduced biological compensation
mutation results in altered transcript which is degraded by nonsense mediated decay
mRNA fragments are bound by Upf3a and transported into the nucleus
Upf3a rectruits protein compflex with chromatin modification capability and binds to gene with sequence similarity via mRNA fragment
chromatin modification to upregulate related gene with sequence similarity
Last changed2 months ago
