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Genetic Engineering (BS3530)
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Word-for-Word Lecture notes for BS3530 at RHUL
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Lecture OnHow to design a eukaryotic (mammalian expression vector
- Concept of mammalian vecto
- Eukaryotic transcription - the basic component
- Translational enhancement: Kozak, codon optimisation
- Mammalian cell lines for gene expression
- Promoter sequences for mammalian cells
- Constitutive/ubiquitous (CMV)
- Tissue specific
- Tumour specific
- Inducible (tet)
- LCRs (barriers to gene expression)
Lecture Notes
- Describe the basic components of a mammalian expression vector
Expression Vectors
Cannot use a prokaryotic system as they may be unstable/inactive, may be subject to toxic/
pyrogenic compound contamination, and have insufficient post-translational modifications.
The goal is to produce a functional eukaryotic protein. This requires: (bacterial) backbone
plasmid, eukaryotic gene expression sequences (promoter), an open reading frame (cDNA/gene
of interest), (mini-intron between promoter and ORF), (selectable marker gene), (eukaryotic
origin of replication).
Cloning in a selectable marker = cloning in a cassette encoding a promoter, the selectable
marker and a terminator. If bacterial backbone not applicable can use restriction enzymes to
remove sections of plasmid.
Promoters are required for transcription initiation, and contain various elements composed
of binding sites for transcription factors.
Enhancers increase transcription levels and may not need to be located directly upstream of
a coding sequence.
Transcription termination sequences include the polyadenylation signal. Polyadenylation
stabilises the RNA and favours export to cytoplasm.
Open reading frame(gene of interest) - May encode any protein that is needed in
biotechnology or research, e.g. blood clotting factor VII
Translation enhancement: Kozak sequences are located around starting ATG with canonical
sequence -gccRccAUGG- (R = purine), these are translation enhancers rather than ribosome
binding sites
Translation enhancement through gene (“codon”) optimisation
- DNA code is redundant → several codons for the same amino aci
- tRNAs for the codons encoding the same amino acid have different frequencies in various
organisms
- Codon optimisation = use only codons corresponding to most abundant tRNAs in species of
interest → faster translation and increased yield
- Further improvements obtained by removal of sequences detrimental to transgenic
expression: repeats, cryptic splicing signals...
Gene expression levels can be improved through the manipulation of cis-acting (altering
sequences present in same molecule as expression cassette) regulatory elements (promoters,
enhancers, Kozak sequence) and optimisation of the coding sequence
Mammalian cell lines as expression system
Most common cell lines
1. CHO, chinese hamster ovary. Most used in biotech: growth in suspension, high protein
yield, refractory to human viruses.
2. HEK293T, human embryonic kidney cells transformed with Adenovirus 5 and Simian Virus 40
(SV40) large T-antigen. Allows plasmid replication. Very easy to transfect. Viral vector
production.
3. COS, simian kidney cells transformed with SV40 large T-antigen. Allows plasmid
replication. Virus biology and production studies.
May be stably or transiently transfected.
e
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,Typical Regulated Gene
- Typical protein coding mammalian gene bears multiple regulatory regions including one or
more enhancers, and a promoter driving expression of a gene composed of multiple exons,
introns, and a signal for polyadenylation.
- Enhancers are located usually 2–100 kb upstream or downstream from the promoter,
sometimes within introns but occasionally downstream of the poly(A) site.
Promoters are subdivided into proximal and core promoters.
- The proximal promoter is located within 2 kb upstream of the transcription start site.
- The core promoter is ∼80–200 bp in length and encompasses the transcription start site.
Proximal promoters and enhancers contain specific DNA sequence motifs, which bind
combinatorial arrays of sequence-specific transcriptional activators that, in turn, recruit
co-activators such as Mediator or EP300.
The core promoter is composed of different combinations of a series of DNA elements
including the TATA box, the INR, the DPE, and others. These DNA elements collectively bind
to TBP and the TAFs, which tether the other general transcription factors, Mediator and pol
II, at the start site. This complex of GTFs, pol II, and Mediator is commonly termed the pol
II PIC. Chromatin loops form between the proteins bound at the enhancer and promoter during
gene activation. The gene and its regulatory elements are often flanked by boundary elements
or insulators, which in mammals typically bind to the sequence-specific factor CCCTC-binding
factor (CTCF). CTCF recruits the Cohesin complex, which catalyses the formation of a
chromatin loop encompassing the gene, the promoter, and its enhancers. This loop spatially
sequesters or insulates the gene and its regulatory elements, preventing its enhancers from
acting on the promoters of flanking genes and vice versa
Essential Readin
Addgene: Promoters
A promoter is a region of DNA where transcription of a gene is initiated → vital component
of expression vectors as they control binding of RNA polymerase to DNA. Promoters are about
100-1000 base pairs long and are typically upstream (5’) of the sense or coding strand of
the transcribed gene.
Coding strand - DNA strand that encodes codons and whose sequence corresponds to the mRNA
transcript produced
Antisense strand - referred to as the template or non-coding strand, as this is transcribed
by RNA polymerase.
DNA sequences called response elements are located within promoter regions, and they provide
a stable binding site for RNA polymerase and transcription factors. Promoter binding is very
different in bacteria compared to eukaryotes. In bacteria, the core RNA polymerase requires
an associated sigma factor for promoter recognition and binding. Eukaryotes require a
minimum of seven transcription factors in order for RNA polymerase II to bind to a promoter.
Promoters are controlled by various DNA regulatory sequences including enhancers, boundary
elements, insulators, and silencers.
Promoter regions:
1. Core promoter → located most proximal to the start codon and contains the RNA polymerase
binding site, TATA box, and transcription start site (TSS). RNA polymerase will bind to
this core promoter region stably and transcription of the template strand can initiate.
The TATA box is a DNA sequence (5’-TATAAA-3’) within the core promoter region where
general transcription factor proteins and histones can bind. Histone binding prevents the
initiation of transcription whereas transcription factors promote the initiation of
transcription. The most 3' portion (closest to the gene's start codon) of the core
promoter is the TSS which is where transcription actually begins. Only eukaryotes and
archaea, however, contain this TATA box. Most prokaryotes contain a sequence thought to
be functionally equivalent called the Pribnow box which usually consists of the six
nucleotides, TATAAT
2. Proximal promoter → further upstream from the core promoter is the proximal promoter
which contains many primary regulatory elements. The proximal promoter is found
approximately 250 base pairs upstream from the TSS and it is the site where general
transcription factors bind
3. Distal promoter → upstream of the proximal promoter and contains transcription factor
binding sites, but mostly contains regulatory elements
https://blog.addgene.org/plasmids-101-the-promoter-region
, TATA
Binding Protein
Pol II core promoter
Core promoter: minimal set of sequence elements required for accurate transcription
initiation by the Pol II machinery, as measured in vitro.
•40-60 nucleotide long extending either upstream or downstream of the transcription
start site. Contains a combination of some of these elements below but not all together.
•These elements are required to bind the general transcription factors, TFIIB, TBP, TFIID
•BRE: TFIIB recognition element
•TATA box
•Inr: initiator element
•DPE: downstream promoter element
•DCE: downstream core element
•MTE: motif ten element not shown, but upstream of DPE
•Regulatory sequences: located outside the core promoter and can be in large distance
These are required for efficient transcription in vivo.
•Categories: promoter proximal elements, upstream activator sequences (UAS),
enhancers, silencers, boundary elements, insulators.
•These elements bind regulatory proteins (activators and repressors)
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