Genetic Engineering and Gene Knockout

14 Questions

What is the primary purpose of adding adaptors during the RNA sequencing process?

To capture fragments containing exons

What is the function of the kanMX gene in targeted mutagenesis?

It provides a selective marker for the knockout construct

What is the result of targeted mutagenesis by homologous recombination in yeast?

A null mutant with a knockout of specific genes

What is the purpose of ES cells in targeted mutagenesis in mouse?

To facilitate homologous recombination

What is the estimated size of the final data generated from Whole-Genome-Sequencing?

~10 GB

What is the main function of the CRISPR-Cas9 system in bacteria?

To recognize and digest foreign DNA

What is the role of PAM (protospacer adjacent motifs) in the CRISPR-Cas9 system?

To guide crRNA and tracrRNA to the gene of interest

What is the result of using RNA interference (RNAi) in gene regulation?

The RNA expressed by the gene of interest is only degraded

What is the difference between NHEJ and HDR in the CRISPR-Cas9 system?

NHEJ results in small insertions or deletions, while HDR results in precise changes

What is an advantage of using the CRISPR-Cas9 system for genome editing?

It can edit multiple genes in one step

What is the primary function of restriction enzymes like EcoRI in bacteria?

To defend against invading viruses

What is the purpose of genetic complementation analysis in yeast?

To identify recessive mutations in the same or different genes

What is the result of combining a DNA fragment with a vector in DNA cloning?

Recombinant DNA

What is the purpose of using a plasmid as a vector in DNA cloning?

To provide sequences for maintaining the vector in the host

Study Notes

Gene Knockout and Genome Editing

Knockout mouse: a mouse in which a specific gene is knocked out in all tissues
ES cells: Embryonic Stem cells used to create knockout mice
Knockout construct can insert at the site of interest or somewhere else in the nuclear genome, which can be determined by ganciclovir sensitivity
LoxP-Cre recombination can be used to knockout specific genes in specific cells or tissues

CRISPR-Cas System

clusters of regularly-interspaced short palindromic repeats (CRISPR) were first observed in bacteria to recognize and digest foreign DNA
CAS: CRISPR-associated proteins, such as nuclease Cas9, used to digest DNA
CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) are used to recognize specific DNA regions
PAM (protospacer adjacent motifs) are important for guiding crRNA and tracrRNA to the gene of interest
NHEJ (Non-homologous end-joining) and HDR (Homology Directed Repair) are two repair pathways used to repair double-stranded breaks
Applications of CRISPR-Cas9 include genome editing, gene regulation, and future directions in human therapeutics and ecological engineering

Gene Inactivation

RNA interference (RNAi) using siRNA (small interfering RNA) can be used to inactivate genes by degrading specific RNA
Sequencing of DNA can be done using conventional sequencing, whole-genome sequencing, whole-exome sequencing, and transcriptome sequencing

Genetic Analysis

Targeted mutagenesis by homologous recombination can be used to knockout specific genes in yeast and mouse
Genetic complementation analysis can be used to determine whether recessive mutations are in the same or different genes
Analysis of double mutants can reveal the order of steps in biosynthetic pathways and interacting or redundant proteins

DNA Cloning

DNA cloning involves the replication of recombinant DNA within host cells
Isolation, sequencing, and manipulation of purified DNA fragments can be done using DNA cloning
A plasmid (vector) contains sequences to maintain it in the host, such as replication origin and selection marker gene
Restriction enzymes, such as EcoRI, can be used to cleave palindromic sequences and create recombinant DNA