Advanced Molecular Biology

Features of Eukaryotic Gene Expression

E• ori is useful for eukaryotic replication (not always possible).

euk• SMG: selectable marker gene under the control of eukaryotic promoter, polyadenylation, and terminalsequence.

• Foreign DNA is cloned into the MCS and is under the control of eukaryotic promoter, polyadenylation, andtermination sequence.

• An intron enhances the production of heterologous protein.

Why an intron close to the site of expression of the POI? Splicing needed to export the mRNA from thenucleus to the cytoplasm, and this enhances the production of heterologous proteins.

Chromatin insulator: to block the spreading of the heterochromatin.

Features for Efficient Gene Expression in Mammals

• For the best results, a gene of interest must be equipped with translation control sequences.

• A gene of interest can be fitted with various sequences that enhance translation and facilitate bothsecretion and purification.

• A Kozak sequence, specific

1. The sequence surrounding the AUG start codon, signal sequence, protein affinity tag for purification, proteolytic cleavage site, and stop codon.
2. The 5' and 3' UTR increase the efficiency of translation and contribute to mRNA stability.
3. Addition of tags to the recombinant protein: in frame (N-ter or C-ter) insertion of a short sequence coding for few amino acids (for example 6His). Insertion of a sequence recognized by a protease for tag removal.
4. Promoters for mammalian expression:
   • Usually constitutive promoters for high expression are used, such as CMV and SV40 viral promoters.
   • Also use enhancers to have a very high expression of the POI.
   • Possible to use non-viral promoters, but they have to be strong, such as the EF-1 promoter.
   • The system use the endogenous subset of TFs able to recognize viral promoters/enhancers or very active endogenous promoters.
5. Often needed a regulated expression (turn-on/turn-off): exploit RE (response elements) that can be placed upstream a core

promoter (ex. hormone RE, antibiotic RE...); sometimes those RE are not commonly expressed in mammalian cells so TFs able to bind to the RE activating transcription are needed: canonical TFs + chimeric TFs (even bacterial/eukaryotic chimeric TFs). An example is the tetracycline regulation of expression system.

TetOn/TetOff system

The E. coli tet operon was first identified as a transposon (Tn10) that confers resistance to the antibiotic tetracycline. The TetR protein, in a similar fashion to the lac repressor protein (LacI), binds to the operator of the tetracycline-resistance operon and prevents RNA pol from initiating transcription. It binds DNA (on tetO) in the absence of tetracycline, inhibiting transcription. When the antibiotic is present, TetR is no more able to bind the DNA and transcription starts.

There are mutants of the tet repressor unable to bind DNA, unless tetracycline is present. In this case, in the absence of tetracycline is unable to bind DNA and transcription is active.

whereas in the presence of tetracycline it becomes able to bind DNA and transcription is inhibited.

The TetOn/TetOff system is based on a chimeric transcription factor consisting of:

• The bacterial tetracycline repressor (TetR): mediates DNA and effector binding as well as dimerization.
• An eukaryotic transcriptional regulatory domain fused to TetR which activates tTA (original TetR) and rtTA (mutated TetR) or silences tTS (original TetR) expression.
• tTS binds to operator in the absence of tet (tetR + a silencing domain). So without tetracycline the expression is off, adding the antibiotic the expression is on.

The chimeric transcription factor acts by binding to a Tet-regulatable promoter containing several TetR-specific binding sites (tetO) that form a tetracycline response element (TRE) placed upstream of a transcription initiation site (core promoter).

Tet-Off activates transgene expression in the absence of effector, Tet-On in its presence. tTS represses endogenous or synthetic

activated promoters in the absence of effector.

Advantages:

• Extremely tight on/off regulation (low background).
• No pleiotropic effects (system dedicated only to our protein expression).
• High indelibility and fast response time.
• High absolute expression levels.
• Well characterized and inexpensive inducer.

Production of protein complexes: recombinant proteins with quaternary structure

Two-vectors expression system: 2 plasmids, one encode for A and one for B; production of the two subunits A and B that than assemble together in the final protein.

One-vector/one promoter expression system: the same vector encode for both A and B, which are under the control of the same promoter.

One-vector/two promoters expression system: the same vector encode for both A and B, which are under the control of two different promoters.

Mutagenesis

Why is mutagenesis needed?

cDNA of the target protein needs to be modify for cloning purposes, increasing transcription

and/or translation efficiency, allowing easy purification.

Native proteins are not well suited for industrial application.

Native proteins are not optimized for medicinal purposes.

Applications of mutagenesis:

• Adding suitable restriction sites for cloning.
• Adding promoter region, RBS, STOP codon, signal peptide.
• Changing rare codons.
• Allowing tag addition.
• Generating chimeric proteins.
• Increasing the efficiency of enzyme-catalyzed reactions.
• Eliminating the need for cofactor in enzymatic reaction.
• Changing substrate binding site to increase specificity.
• Changing the pH stability/thermal stability.
• Increasing protein resistance to proteases (purification).
• Detoxifying.
• Probing mechanisms of protein function/protein structure/protein interaction.

16· PCR-mediated insertional mutagenesis

Terminal sites: addition of restriction sites, promoters, RBS, start codons, stop codons, termination sequences.

Gene fusion:

heteroduplexes fill-in step, always check reading frame.

This method is useful for the construction of chimeric genes.

Domain swapping:

asymmetric PCR to obtain a mega-primer, preferentially amplify one strand more than the other adding a greater excess of one primer for the chosen strand. In the examples, the forward primer is the one in excess to obtain a mega-primer.

PCR-mediated deletion mutagenesis

Terminal deletion: Oligonucleotide design allows precision in deletion positions.

Domain deletion: asymmetric PCRs. Always check reading frame.

Site specific mutagenesis

PCR-mediated introduction of point mutagenesis (mutagenic primers)

Primer extension methods.

Drawbacks:

• Both mutant and wild type versions of the gene are produced following transformation.
• Lots of screening required.
• Requires single-stranded, circular template DNA.

Alternative primer extension mutagenesis techniques:

QuikChage protocol can use plasmid, easy screen because no wild-type DNA will be

present.· Random mutagenesis

The mutations are randomly inserted in the DNA by techniques in which the DNA replication system is altered.

The gene that encodes a protein (starting sequence) is modified by introducing relatively random mutations(substitutions, deletions, and insertions) at random positions.

Chemical and physical mutagens: like alkylating agents or UV irradiation, the damaged DNA is incorrectlyv replicated or repaired, causing the mutation.

Mutator strains: bacteria that lack one or more mechanisms of DNA repair resulting in higher mutationv rates and thus a greater rate of accumulation of mutations compared to normal strains.

These two methods have the disadvantage of introducing mutations in an indiscriminate manner along the entirebacterial genome (survival, expression and translation) but also the vector carrying the transgene (expression andtranslation, replication). Furthermore, the second method (mutator strains) is slow. The level of mutagenesis islinked to

The length of time the recombinant gene spends in the mutator strain. 19Mutagenic PCR is the most widely used method.v Error-Prone PCR. The mutations are introduced under conditions that cause an increase in the speedØ by which the DNA polymerase generates errors. Mutagenic rate of ≈1 nt/kb.The increase in errors during PCR can be obtained in various ways:

• Highly error-prone Taq polymerase, a pol with lower replicative fidelity (for example lacking the§ proof reading activity)
• Addition of small amount of Mn instead of only Mg .§ 2+ 2+
• Different concentrations of dNTPs: limiting concentration of one of the four.§
• DNTP analogs. Mutagenic dNTP analogues are incorporated into the amplified DNA fragment. TheseØ analogues allow mispairing with the natural nt and are eliminated at the second step of PCR which is conducted only in the presence of the 4 natural dNTPs. Mutagenic rate: 1 every 5 nt.

14/11 Transgenic animalsTransgenesis: introduction of foreign genes

Transgenesis is the process of introducing additional or altered forms of an endogenous gene into an organism. It typically does not result in the replacement of endogenous genes, but rather adds the new gene and integrates it into the genome. Often, multiple copies of the gene of interest are introduced. Transgenesis is useful for studying the function of a gene and altering its expression. It can involve introducing a mutated copy of an endogenous gene to study the role of the mutation and how it affects the activity of the endogenous copy. Transgenesis is also useful for producing proteins of interest, such as drugs.

Methods of producing transgenic animals, specifically mice, include the pronucleus method. This involves directly injecting the desired gene (vector with the cDNA coding for the protein + regulatory regions) into the pronucleus of a fertilized mouse egg. A female animal is super ovulated, and eggs are collected and fertilized in vitro. The transgene-containing solution is then injected into the male pronucleus using a micropipette. Eggs with the transgene are kept...

overnight in an incubator to develop to a 2 cell stage. The eggs are then implanted into the uterus of a pseudo-pregnant female, a female which has been mated with a vasectomized male the previous night.

ES cell method

Collecting ES cells from inner mass of blastocyst. Transfer (ex. electroporation, retro-virus...) the transgene in the ES cells. Selection of the cell for the acquisition of the transgene (selection marker in the vector). The positive clone would be planted in a new inner mass. The blastocyst will be planted in pseudo-pregnant female mice. About 1/3 of the implanted blastocysts survive. No more than 10-20% of the offspring will express the transgene.

Selection of chimeric animals to have the homozygous ones for the transgene. This one is a longer procedure.

Screening of chimeric animals: just the ones with the transgene in germ lines can transmit it to the progeny. These animals are heterozygotes, so a cross between the heterozygous progeny has to be done to