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Which of the following are characteristic features of a plasmid?

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Contains an origin of replication

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To avoid off-target effects by CRISPR/Cas9 the Fok1 endonuclease is fused to -----.

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dCas9

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Fusion of deactivated Cas9 to Fok1 endonuclease reduces off-target effects because ----.

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two different but neighboring DNA sequences are targeted

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which characteristics do F-plasmids confer to the host bacterium?

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Conjugative ability

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Which of the following is an example of head-and-tail bacteriophage?

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Lambda Phage

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The cycle which leads to lysis of the bacteria and release of phage particles is called ...

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Lytic

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Pilus helps in the transfer of DNA through a process of ...?

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Conjugation

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Transgenesis of mice by genetic engineering involves -----.

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Recombinant DNA

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Which features below are required for efficient homologous recombination?

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Homologous sequences

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What does DSB stand for in the context of homologous recombination?

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DNA single break

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Zinc-finger nuclease and TALENs can perform -----.

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DNA cuts

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where can sticky ends be found?

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12 nucleotide stetch in lamda phage

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Q:

Which of the following are characteristic features of a plasmid?

A:

Contains an origin of replication

Q:

To avoid off-target effects by CRISPR/Cas9 the Fok1 endonuclease is fused to -----.

A:

dCas9

Q:

Fusion of deactivated Cas9 to Fok1 endonuclease reduces off-target effects because ----.

A:

two different but neighboring DNA sequences are targeted

Q:

which characteristics do F-plasmids confer to the host bacterium?

A:

Conjugative ability

Q:

Which of the following is an example of head-and-tail bacteriophage?

A:

Lambda Phage

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Q:

The cycle which leads to lysis of the bacteria and release of phage particles is called ...

A:

Lytic

Q:

Pilus helps in the transfer of DNA through a process of ...?

A:

Conjugation

Q:

Transgenesis of mice by genetic engineering involves -----.

A:

Recombinant DNA

Q:

Which features below are required for efficient homologous recombination?

A:

Homologous sequences

Q:

What does DSB stand for in the context of homologous recombination?

A:

DNA single break

Q:

Zinc-finger nuclease and TALENs can perform -----.

A:

DNA cuts

Q:

where can sticky ends be found?

A:

12 nucleotide stetch in lamda phage

Genetic Engineering

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Eine der Genetic Engineering Zusammenfassungen auf StudySmarter | Hochschule Bonn-Rhein-Sieg

Lecture 1

Genetic Engineering starts with cloning. 

Cloning: 

  1. Isolation of DNA
  2. Introduce DNA fragment into bacteria
  3. Amplification = Cloning



Use of Genetic Engineering:

Generation of Genetically modified organisms (GMOs)

  • living organisms whose genetic material has been modified through genetic engineering

Example:  

  • AquaAdvantage Salmon: first approved GMO
    • produces growth hormones all year
  • can occur naturally: sweet potatoes (transgenic food)

circa 8000 BCE Humans:

use selective breeding to generate plants and animals with more desirable traits.


GMOs with Gene Drive: 

Uses CRISPR to insert and spread a genetic modification through a population at higher than normal rates of inheritance.

offspring of gene drive animal will inherit the drive on one chromosome and a normal gene from its other parent.





Genetic Engineering for Reprogramming

Direct reprogramming: reprogramming of one differentiated cell into another differentiated cell.

standard reprogramming: reprogramming of mature cells into induced pluripotent stem cells (iPSCs)

Reprogramming could be used for Regenerative Medicine

Yamanaka factors

bind to the promoter before the stem cell gene. 

mRNA can be produced and from there stem cell proteins can be produced.


Barrier Genes 

induce repressive chromatin. they are a problem for reprogramming because the transcription factors cannot bind

Caenorhabditis elegans (C.elegans)


70% of all 20.000 C.elegans genes have Human homologs: 

  • Apoptosis & Development
  • GFP
  • RNA interference    
  • miRNA

C.elegans is transparent.

Genetic Engineering to visualize tissues.

Genetic Engineering to inactivate genes

Lecture 2

Plasmids: independent genetic elements found in bacterial cells.



Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell. they carry one or more genes. The genes are responsible for useful characteristics displayed by the host bacterium.



Phages are viruses that specifically infect bacteria.


Phages are simple in structure, consisting merely of a DNA molecule carrying a number of genes surrounded by a protective coat or capsid made up of protein molecules.

The infection cycle of bacteriophage λ















Basic cloning procedure requires a Vector = Plasmid

  1. A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a recombinant DNA molecule.
  2. the vector transports the gene into the host cell, which is usually a bacterium, although other types of living cells can be used.
  3. Within the host cell, the vector multiplies, producing numerous identical copies, not only of itself but also of the gene that it carries.
  4. When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place.
  5. After a large number of cell divisions, a colony, or clone, of identical host cells is produced. Each cell in the clone contains one r more copies of the recombinant DNA molecule. The gene carried by the recombinant molecule is now said to be cloned.






Antibiotic resistance as a selectable marker for a plasmid


In the laboratory, antibiotic resistance is often used as a selectable marker to ensure that bacteria in culture contain a particular plasmid.

RP4 carries genes for resistance to ampicillin, tetracycline, and kanamycin. only those E.coli cells that contain RP4 ( or a related plasmid) are able to survive and grow in a medium that contains toxic amounts of one or more of these antibiotics.










Bacteria need time for synthesis of antibiotic-resistance enzymes

The resistance to the antibiotic is not due merely to the presence of the plasmid in the transformed cells. the resistance gene on the plasmid must also be expressed, s that the enzyme which detoxifies the antibiotic is synthesized. Although the expression of the resistance gene begins immediately after transformation, it takes a few minutes before the cell contains enough of the enzyme to be able to withstand the toxic effects of the antibiotic. For this reason, the transformed bacteria should not be plated onto the selective medium immediately after a heat-shock treatment, but should first be placed in a small volume of the liquid medium, in the absence of the antibiotic, and incubated for a short time.

Antibiotic selection: Ampicillin

Antibiotic selection: Tetracycline & Kanamycin

The antibiotic selection allows the growth of selected plasmids

Cloning vectors were derived from combining natural plasmids

The ampR gene was obtained from Tn3, a type of transposable element carried by the R1 plasmid. the tetR gene was excised from pSC101 by treatment with EcoR1 in a low-salt solution, which decreases the specific of the enzyme so that, as well as cutting at its standard GAATTC recognition sequence, it also cuts at related sequence such as TAATTC. This is called star activity, and the related sequences are referred to as EcoR1* sites.

Plasmid nomenclature

The name ‘pBR322’ conforms with the standard rules for vector nomenclature:

‘p’ indicates that this is indeed a plasmid.

‘BR’ identifies the laboratory in which the vector was originally constructed (BR stands for Bolivar and Rodriguez, the two investigators who developed pBR322).

‘322’ distinguishes this plasmid from others developed in the same laboratory (there are also plasmids called pBR325, pBR327, pBR328, etc.).

Transformation


E.coli cells that had been soaked in an ice-cold salt solution were more efficient at DNA uptake than unsoaked cells. A solution of 50mM calcium chloride (CaCl2) is traditionally used for this purpose. When DNA is added to treated cells it remains attached to the ell exterior and is not at this stage transported into the cytoplasm. the actual movement of DNA into competent cells is stimulated by briefly raising the temperature to 42°C.

The manipulations that result in a recombinant DNA molecule can only rarely be controlled to the extent that no other DNA molecules are present at the end of the procedure.  The ligation mixture may contain, in addition to the desired recombinant molecule, any number of the following: 

  • Unligated vector molecules
  • unligated DNA fragments
  • Vector molecules that have recircularized without new DNA being inserted (´self-ligated´ vector)
  • Recombinant DNA molecule that carries the wrong inserted DNA fragment.

Unligated molecules rarely cause a problem because even though they may be taken up by bacterial cells, only under exceptional circumstances will they be replicated. Self-ligated vector molecules and incorrect recombinant plasmids are ore important because they are replicated just as efficiently as the desired molecule. Purification of the desired molecule can still be achieved through cloning because it is extremely unusual for anyone cell to take up more than one DNA molecule.

b-galactosidase is expressed when the repressor protein becomes inactivated

The lacZ gene in plasmids produces β-galactosidase.

Agar plate containing X-gal (5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside) à blue color IPTG (Isopropyl β-d-1-thiogalactopyranoside) à mimics allolactose à inactivates lac repressor

white colonies ace additional gene

Plasmid for in vitro transcription of cloned DNA: pGEM3Z







F-plasmid: conjugation
















Conjugation allows DNA transfer

Conjugative F-plasmid can integrate into the bacterial chromosome

Cointegrate formed between two plasmids by recombination between homologous sequences

Plasmid amplification

The aim of amplification is to increase the copy number of a plasmid. Some multicopy plasmids have the useful property of being able to replicate in the absence of protein synthesis. After a satisfactory cell density has been reached, an inhibitor of protein synthesis is added and the culture incubated for a further 12h. The plasmid molecules continue to replicate, but the chromosome replication and cell division are blocked.

Basic steps in preparation of total cell DNA from a culture of bacteria


After cell lysis, all other material than DNA needs to be removed

Removal of protein contaminants by phenol extraction

Deprotoneinize a cell extract by adding phenol or a 1:1 phenol and chloroform mixture. they precipitate proteins but leave the nucleic acids in an aqueous solution. After centrifugation, the precipitates are left as a white coagulated mass.  

DNA purification by ion-exchange chromatography

Alternative methods of using silica to purify DNA from a cell extract

Plasmids do not sediment to the pellet-like larger DNA fragments

Controlled lysis is performed. Treatment with EDTA and lysozyme carried out in presence of sucrose. Sucrose prevents cells from bursting immediatley. Spheroblasts are formed. Spheroblasts = cells with partially degraded cell walls retaining an intact cytoplasmic membrane. Cell lysis induced by adding a nonionic detergent. Causes very little breakage of bacterial DNA → centrifugation results in cleared lysat consisting almost entirely of plasmid DNA.

Conformations of circular double-stranded Plasmid DNA.

Most plasmids exit in the cell as supercoiled molecules. can be aintained only if both polynucleotide strands are intact → covalently closed-circular (ccc) DNA.
if one polynucleotide strand is broken, the double helix reverts to its normal relaxed state and the plasmid takes on the open-circular (oc) conformation.

A Basic Cloning Example

Restriction Enzymes Cleave DNA at a Specific Sequence

Restriction endonuclease cleaves Phage DNA but not bacterial DNA

Restriction occurs because the bacterium produces an enzyme that degrades the phage DNA before it has time ti replicate and direct the synthesis of new phage particles. The bacterial DNA is protected from attack because it carries additional methyl groups that block the degradative enzyme action. these degradative enzymes are called restriction endonucleases and are synthesized by many species of bacteria. Three different types of endonucleases are recognized. Types 1 and 3 are complex and have a limited role in genetic engineering. Type 2 restriction endonucleases are the cutting enzymes that are so important in gene cloning.

Restriction enzymes: endonuclease versus exonuclease

Exonucleases: remove nucleotides one at a time from the end of a DNA molecule.

Endonucleases: break internal phosphodiester bond within a DNA molecule.

Recognition sequences for some restriction endonucleases

Palindromic recognition sequences

the main distinction between different exonucleases lies in the number of strands that are degraded when a double-stranded molecule is attacked.

Lecture 3

Restriction Enzymes from different bacteria cleave DNA at a Specific ‘palindromic’ Sequence

A multiple cloning site (MCS) contains several Restriction Enzyme sites.

Restriction of the λ DNA molecule

the number of recognition sequences for a particular restriction endonuclease in a DNA molecule of known length can be calculated mathematically.  Many of these recognition sites occur less frequently. Restriction sites are generally not evenly spaced out along a DNA molecule.  Math may give an idea of how many restriction sites to expect in a given DNA molecule, only experimental analysis can provide the true picture.

Before adding the enzymes, the solution containing the DNA must be adjusted to provide the correct conditions to ensure maximal activity of the enzyme. It is also advisable to add a reducing agent, such as dithiothreitol (DTT), which stabilizes the enzyme and prevents its inactivation. Providing the correct conditions for the enzymes is very important.

How can we separate different DNA Fragments?

Each vector molecule must be cleaved at a single position, to open up the circle so that new DNA can be inserted. A molecule that is cut more than once will be broken into two or more fragments and will be useless as a cloning vector. Each vector must be cut at exactly the same position on the circle. 

Often, it is necessary to cleave the DNA that is to be cloned. This is necessary for two reasons:

  1. if the aim is to clone a single gene, then that gene will have to be cut out of the large DNA molecules
  2. large DNA molecules must be broken to produce small fragments that can be carried by the vector.

Ligation: connecting DNA fragments

The enzyme used in genetic engineering is usually purified from E. coli bacteria that have been infected with T4 phage = T4 DNA Ligase can join blunt and sticky ends.

Restriction and ligation to generate a recombinant plasmid

DNA topoisomerase is useful to ligate PCR products into a special plasmid with T-overhangs.

Special cloning vector carrying thymidine overhangs which can be ligated to a PCR product. they are prepared by restricting a standard vector at a blunt-end site, and then treating with Taq polymerase in the presence of just 2´-deoxythymidine 5´-triphosphate (dTTP).

How can we select or identify just one gene?

DNA Polymerase Adds Nucleotides to the 3' End of a DNA Chain

Basic Principles of PCR

25 rounds of amplification produce 225 copies

Hybridization of the oligonucleotide primers to the template DNA

2nd cycle of a PCR produces the ‘short’ products

Primer design is important for successful PCR

Primers must correspond with the sequence flanking the target region on the template molecule. Each primer must be complementary to its template strand in order for hybridization to occur, and the 3´ends of the hybridized primers should point towards one another.

The lengths of the primers are critical for PCR specificity

If the primers are too short they might hybridize to non-target sites and give undesired amplification products. the length of the primer influences the rate at which it hybridizes to the template DNA, with long primers hybridizing at a slower rate.

A typical temperature profile for a PCR

Denaturation: beaks the base pairs and releases single-stranded DNA to act as templates in the next round of DNA synthesis.
Annealing: primers attach to the template
Extension: DNA synthesis occurs.

The melting temperature or Tm

Tm = (4×[G+C]) + (2×[A+T])°C

The annealing temperature during PCR should be 3-5°C below Tm.

Gradient PCR can solve issues with different primer Tm. If primer Tm is different for forward and reverse by more than 10°C → designing new primer(s) is often recommended.

After the PCR….

Restriction fragment length polymorphism (RFLP) analysis.

TA cloning

Primers containing restriction sites. After PCR, the products are treated with the restriction endonuclease, leaving sticky-ended fragments that can be ligated efficiently into a standard cloning vector. the restriction site can be included within a short extension at the 5´end of each primer. these extensions cannot hybridize to the template molecule, but they are copied during PCR, resulting in PCR products that carry terminal restriction sites.

PCR is also used for ‘Sanger’ Sequencing

Sanger-Sequencing

A short oligonucleotide is annealed to the template DNA, this oligonucleotide subsequently acting as the primer for synthesis of a new DNA strand that is complementary to the template. to stop the continuous addition of new nucleotides a small amount of each of four dideoxynucleotides(ddNTPs) is added to the reaction.

Problems with the error rate of Taq polymerase

Proofreading Polymerases solve the problem…

a)The 5´→ 3´actiity has an important role in DNA repair in the cell, as it enables the polymerase to replace a damaged DNA strand.
b) the 3´→ 5´activity also has an important role in the cell, as it allows the polymerase to correct its own mistakes, by reversing and replacing a nucleotide that has been added in error. This is called proofreading. During DNA sequencing, this activity can result in the removal of a dideoxynucleotide that has just been added to the newly synthesized strand, so that chain termination does not occur.
Pfu DNA Polymerase was isolated from Pyrococcus furiosus. It possesses 3´→5´ exonuclease (proofreading) acCvity. Pfu DNA Polymerase- generated PCR fragments are blunt-ended. T/A cloning is not possible.AccuTaq™ LA DNA Polymerase is uClized to amplify DNA sequences including genomic targets larger than 20 kb, as a result of a mixture of high-quality Taq polymerase with a proofreading polymerase. T/A cloning is possible.


The Expand High Fidelity PCR System is composed of an enzyme mix containing thermostable Taq DNA polymerase and a thermostable DNA polymerase with proofreading acCvity. T/A cloning is possible.



Proofreading Polymerases can not be used for Sequencing

Reverse Transcriptase – a very special DNA Polymerase

Reverse Transcriptase: mRNA can be cloned as complementary DNA (cDNA)

Transcript analysis by PCR: Reverse Transcription can also be used to identify transcribed genes

Each cell contains the same complement of genes, but in different cell types, different sets o genes are switched on, while others are silent. Only those genes that are being expressed are transcribed into mRNA.

Transcript analysis by PCR: Reverse Transcription for Rapid Amplification of cDNA Ends (RACE)

Identify 5´and 3´termini of RNA molecules and can be used to map the ends of transcripts. Primer specific for an internal region of the RNA molecule. Primer attaches to the RNA and directs the first, reverse transcriptase-catalyzed stage of the process, during which a single-stranded cDNA is made. As in the primer extension method, the 3´end of the cDNA corresponds with the 5´end of the RNA. Terminal deoxynucleotide transferase is now used to attach a series of A nucleotides to the 3´end of the cDNA, forming the priming site for a second PCR primer, which is made up entirely of Ts and hence anneals to the poly(A) tail created by terminal transferase. Now the standard PCR begins, first converting the single-stranded cDNA into a double-stranded molecule, and then amplifying this molecule as the PCR proceeds. The PCR product is then sequenced to reveal the precise position of the start of the transcript.


PCR to introduce mutations in cloned DNA fragments

Why engineering mutations? Example: to investigate effects of Histone mutations in Cancer

Histone Mutations can cause Brain Cancer!

Histone Mutations cause Cancer by affecting chromatin!

Experimental Histone Mutations to study the cause of Cancer!

reverse transcription of mRNA to cDNA

The general pattern of infection of a bacterial cell by a bacteriophage

The infection cycle of bacteriophage λ and M13

the infection cycle for lambda phage as described earlier.

The M13 DNA is not integrated into the bacterial genome and does not become quiescent. With these phages, cell lysis never occurs, and the infected bacterium can continue to grow and divide, albeit at a slower rate than uninfected cells.

Linear and circular forms of λ DNA

The positions and identities of all of the genes in the λ DNA molecules are known. The molecule on top s linear with two free ends, representing the DNA present in the phage head structure. Consists of two complementary strands of DNA.
The DNA is contained in the polyhedral head structure and the tail serves to attach the phase to the bacterial surface and to inject the DNA into the cell.
At either end of the molecule is a short 12-nucleotide stretch in which the DNA is single-stranded. These two single strands are complementary and so can base pair with one another to form a circular, completely double-stranded molecule.

cos sites are required during replication of λ DNA

The second role of the cos sits comes into play after the prophage has excised from the host genome. At this stage, a large number of new λ DNA molecules are produced by the rolling circle mechanism of replication, in which a continuous DNA strand is ´rolled off´ the template molecule. The result is a catenane consisting of a series of linear λ genomes joined together at the cos sites. The role o the cos sites is now to act as recognition sequences for an endonuclease that cleaves the catenane at the cos sites, producing individual λ genomes. This endonuclease, which is the product of gene A on the DNA molecule, creates the single-stranded sticky ends and also acts in conjunction with other proteins to package each λ genome into a phage head structure.

The M13 DNA molecule is smaller and single-stranded!

  1. Once inside the cell the single-stranded molecule acts as the template for synthesis of a complementary strand, resulting in normal double-stranded DNA.
  2. This molecule is not inserted into the bacterial genome, but instead replicates until over 100 copies are present in the cell.
  3. When the bacterium divides, each daugther cell receives copies of the phage genome, which continues to replicate, thereby maintaining its overall numbers per cell. New phage particles are continuously assembled and released, with about 1000 new phages being produced during each generaion of an infected cell.

Before Transfection of λ cloning vectors, they need to be ‘packed in vitro’

Transfection: phage is involved

The second role of cos sites is rather different, and comes into play after the prophage has excised from the host genome. At this stage a large number of new λ DNA molecules are produced by the rolling circle mechanism of replication, in which a continuouss DNA strand is ´rolled off´the template molecule. The result is a catenane consisting of a series of linear λ genomes joined together at the cos sites. The role of the cos sites is now to act as recognition sequences for an endonuclease that cleaves the catenane at the cos sitees, producing individual λ genomes. This endonuclease, which is the product of gene A on the DNA molecule, creates the single-stranded sticky end, and also acts in conjunction with other proteins to package each λ genome into a phage head structure.

A single-strain packaging system

Synthesis of λ capsid proteins by E. coli strain SMR10, which carries a λ phage that has defective cos sites.

with the single strain system, the defective λ phage carries a mutation in the cos sites, so that these are not recognized by the endonuclease that are normally cleaves the λ catenanes during phage replication. This means that the defective phage cannot replicate, though it does direct the synthesis of all the proteins needed for packaging. These proteins accumulate in the bacterium and can be purified from cultures of E.coli infected with the mutated λ. The protein preparation is then used for in vitro packaging of recombinant λ molecules.

A two-strain packaging system

Synthesis of incomplete sets of λ capsid proteins by E. coli strains BHB2688 and BHB2690.

Neither strain is able to complete an infection cycle in E.coli because, in the absence of hte product of the mutated gene, the complete capsid structure cannot be made. Instead the products of all the other coat protein genes accumulate. An in vitro packaging mix can therefore be prepared by combining lysates of two cultures of cells, one infected with the λ D- strain, the other infected with the E- strain. The mixture now contains all the necessary components for in vitro packaging.
With both systems, the formation of phage particles is achieved simply by mixing the packaging proteins with λ DNA, because assembly of hte particle occurs automatically in the test tubes. The packaged λ DNA is then introduced into E.coli cells simply by adding the assembled phage to the bacterial culture and allowing the normal λ infective process to take place.

Phage infection is visualized as plaques on an agar medium

  1. If infected cell are spread onto a solid agar medium immediatley after addition of the phage particles, or immediatley after transfectin wiht phage DNA, cell lysis can be visualized as plaques on a lawn of bacteria.
  2. Each plaque is a zone of clearing produced as the phages lyse the cells and move on to infect and eventually lyse the neighboring bacteria.
  3. M13 causes a decrease in the growth rate of infected cells, sufficient to produce a zone of relative clearing on a bacterial lawn. Although not true plaques, these zones of clearing are visually identical to normal phage plaques.

M13 Vectors can be used to generate single-stranded versions of cloned DNA

The normal M13 genome is 6.4kb in length, with most of the DNA taken up by ten closely packed genes each essential for the replication of the phage. There is only a single 507-nucleotide intergenic sequence into which new DNA could be inserted without disrupting one of these genes, and this region includes the replication origin which must itself remain intact. This means that there is only limited scope for modifying the M13 genome.

Hybrid plasmid–M13 vectors (Phagemids)

pEMBL8 was made by transferring into pUC9 a 1300bp fragment of the M13 genome. This piece of M13 DNA contains the single sequence recognized by the enzymes that convert the normal double-stranded M13 molecule into single-stranded DNA before the section of new phage particles. This single sequence is still functional even though detached from the rest of the M13 genome, so pEMBL8 molecules are also converted into single-stranded DNA and secreted as defective phage particles.

Identification of recombinant M13 and λ vectors

All M13 cloning vectors, as well as several λ vectors, carry a copy of the lacZ´ gene. The insertion of new DNA into this gene inactivates β-galactosidase synthesis, just as with the plasmid vector pUC8. Recombinants are distinguished by plating cells onto X-gal agar. Plaques comprising normal phages are blue, recombinant plaques are clear.

Identification of recombinant phages: inactivation of the λ cI gene

Several types of λ cloning vectors have unique restriction sites in the cI gene, the insertional inactivation of which causes a change in plaque morphology. Normal plaques appear ´turbid, whereas recombinants with a disrupted cI gene are ´clear´. The difference is readily apparent to the experienced eye.

Selection using the Spi phenotype or on basis of λ genome size


Some λ cloning vectors are designed so that the insertion of new DNA causes a change from Spi+ to Spi-, enabling the recombinants to infect cells that carry P2 prophages. Such cells are used as the host for cloning experiments with these vectors. As only the recombinants are Spi-, only recombinants will dorm plaques.


Regular λ genome has a size limitation to package it into the phage head

The λ DNA molecule can be increased in size by only about 5%, representing the addition of only 3kb of new DNA. If the total size of the molecule is more than 52kb, then it cannot be packaged into the λ head structure and infective phage particles are not formed. This severely limits the size of a DNA fragment that can be inserted into an unmodified λ vector.

Segments of the λ genome can be deleted without impairing viability

Insertion vectors based on λ DNA

  1. With an insertion vector, a large segment f the non-essential region has been deleted, and the two arms ligated together. An insertion vector possesses at least one unique restriction site into which new DNA can be inserted. The size f the DNA fragment that an individual vector can carry depends on the extent to which the non-essential region has been deleted.
  2. λgt10 can carry up to 8kb of new DNA, inserted into a unique EcoRI site located in the cI gene. insertional inactivation of this gene means that recombinants are distinguished as clear rather than turbid plaques.
  3. λZAPII with which insertion up to 10kb DNA into any of six restriction sites within a polylinker inactivates the lacZ´ gene carried by the vector. Recombinants give clear rather than blue plaques on X-gal agar.

Replacement vectors based on λ DNA

Replacement vectors can carry larger pieces of DNA than insertion vectors.

A λ replacement vector has two recognition sites for the restriction endonuclease used for cloning. These sites flank a segment of DNA that is replaced by the DNA to be cloned. Often, the replaceable fragment (called the stuffer fragment) carries additional restriction sites that can be used to cut it up into small pieces, so that its own re-insertion during a cloning experiment is very unlikely.
λDASHII can carry inserted DNA of between 9 and 23kb by replacing a segment flanked by various restriction sites, any of which can be used to remove the stuffer fragment so that DNA fragments with a variety of sticky ends can be cloned. Recombinant selection with λDASHII can be on the basis of size or can utilize the Spi phenotype.

Cloning experiments with λ insertion or replacement vectors

  1. A cloning experiment with a λ vector can proceed along the samelines as wwith a plasmid vector: the λ molecules are restricted, new DNA is added, the mixture is ligated, and the resulting moleules are used to transfect a competent E.coli host. This type of experiment requires that the vector be in its circular form, with the cos sites hydrogen-bonded to each other.
  2. The first improvment would be to use the linear form of the vvector. When the linear form of the vector is digested with the relevant restriction endonuclease, the left and right arms are released as separate fragments. A recombinant molecule can be constructed by mixing together the DNA to be cloned with the vector arms. Ligation results in several molecular arrangements, including catenanes comprisign left arm-DNA.right arm repeated many times.

Long DNA fragments can be cloned using a Cosmid

A cosmid is basically a plasmid that carries a co site. It also needs a selectable marker (such as the ampicillin resistance gene) and a plasmid origin of replication, since cosmid lack the λ genes and so do not prodce plaques. Instead colonies are formed on selective media, just as with a plasmid vector.
A cloning experiment with a cosmid is carried out. The cosmid is opened at its unique restriction site and new DNA fraggments are inserted. These fraggments are usually produced by partial digestion with a restriction endonuclease, as total digestion will almost invariably result in fragents that are too small to be cloned with a cosmid. Ligation is carried out so that catenanes are formed. Provded that the inserted DNA is the correct size, in vitro pakcaging cleaves the cos sites and places the recombinant cosmids into mature phage particles. These λ phages are then used to infect an E.coli culture, alhough of course plaques are not formed. Instead, infected cells are plated onto a selective medium and antibiotic-resistance colonies are grwon. All colonies are recombinants, as non.recombinant linea cosmids are too small to be packaged into λ heads.

Primer with Restriction sites

Errors become much more of a problem if the PCR products re cloned. A seach resulting clone contains multiple copies of a single amplified fragment, the cloned DNA will not necessarily have the same sequence as the original template molecule used in the PCR.

PCR to introduce mutations in DNA fragments

PCR to introduce mutations in cloned DNA fragments

SARS-CoV-2 is an enveloped single-stranded RNA virus

Reverse Transcription + PCR required for Corona-Test

Finding out the transcription start site….

all genes have to be expressed in order to function. The first step in expression is transcription of the gene into a complementary RNA strand. The moleular biologist will want to know wether the transcript is a faithful copyof the gene, or wether segments of the gene are missing from the transcript. These missing pieces are called introns, and considerable interest centers n their structure and possible ffunction. In addtion to introns, the exact locations of the start and end points of transcription are iportant. ost transcripts are copies not only of hte gene itself but also of the nnucleotide regions either side of it.

Other types of ‘Vectors’


Phage infection is visualized as plaques on an agar medium


Preparation of bacteriophage DNA

bacteriophage particles can be obtained in large numbers from the extracellular medium of an infected bacterial culture

bacteriophage particles can be obtained in large numbers from the extracellular medium of an infected bacterial culture.  When such a culture is centrifuged the bacteria are pelleted, leaving the phage particles in suspension. The phage particles are then collected from the suspension and their DNA extracted by a single deproteinization step to remove the phage capsid.

Max. titer that can reasonably be expected for λ is 1010 /ml >> 1010 λ particles will yield only 500 ng of DNA. Large culture volumes, in the range of 500–1000 ml, are therefore needed if substantial quantities of λ DNA are required.

Growth of cultures to obtain a high λ titer

temperature-sensitive (ts) mutation in the cI gene:

  • cI gene is responsible for maintaining the phage in an integrated state
  • ‘ts’ mutation means that at 42°C this gene becomes dysfunctional
In order to obtain a high yield of extracellular λ, the culture must be induced, so that all the bacteria enter the lytic phase of the infection cycle, resulting in cell death and the release of λ particles into the medium. although induction is normally very difficult to control, most laboraatory strains of λ carry a temperature-sensitive (ts) mutation in the cI gene. This is one of the genes responsible for maintaining the phage in an integrated state. If inactivated by a mutation the cI gene no longer functions correctly and the switch to lysis occurs. A culture of E.coli infected with a λ phage carrying the cIts mutation can therefore be induced to produce extracellular phages by transferring from 30°C to 42°C.

Preparation of non-lysogenic λ phages

  1. The key to obtaining a high titre lies in the way in which the culture is grown, and in particular the stage at which the cells are infected by adding phage particles. If ühages are added before the clls are dividing at their maximal rate, then all the cells are lysed very quickly, resulting in a low titre.
  2. if the cell density is tooo high when te phages are added, the culture will never be completely lysed and again the phage titre will be low.
  3. The ideal situation is when the age of the culture an the size of the phage inoculum are balanced such that the culture continues to grow, but eventually all the cells are infected and lysed.

Purification of DNA from λ phage particles

Phage particles are so small that they are pelleted only by very high-speed centrifugation. Hence, the collection of phages is usually achieved by precipitation with polyethylene glycol (PEG) a long-chain polymeric compund which, in the presence of salt, absorbs water, thereby causing macromolecular assemblies such as as phage particles to precipitate. The precipitate can then be collected by centrifugation, and redissolved in a suitably small volume.

Preparation of a gene library

A genomic library is a collection of clones sufficient in number to be likely to contain every single gene present in a particular organism!

Preparation of a gene library in a Cosmid vector

Genomic libraries are prepared by purifying total cell DNA, and then making a partial restriction digest, resulting in fragments that can be cloned into a suitable vector, usually a λ replacement vector, a comid, or possibly a yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1 vector.

Note: There are different types of DNA libraries!!

Differences between genomic DNA versus cDNA libraries

Genomic librarycDNA libraries
it include all possible fragments of DNA from a given cell or organismcDNA library carries only expressed gene sequences.
it is largerIt is smaller
It represents the entire genome of an organism having both coding and no coding regionsIt represents only the expressed part of the genoe and contain only coding sequences called ESTs
Expression of genes taken from genomic library is difficult in prokaryotic system like bacteria due to absence of splicing mechanism.cDNA has only coding sequences therefore can be directly expressed in prokaryotic system.
Vectors used genomic library include plasmid, cosmid, lambda phage, BA and YAC in order to accommodate large fragmentsVectors used cDNA library include plasmid, phagemids, lambda phage etc. to accommodate small fragments as cDNA has no introns.
λ and other high-capacity vectors enable genomic libraries to be constructed

Fosmids are variants of Cosmids but are ‘low-copy’ in E.coli

Some genomic libraries based on Cosmid vectors were unstable. ‘High copy’ of the Cosmid vector in E. coli caused deletions and rearrangements.

→alternative high capacity cloning vector was needed

F-factor origin of replication (from F-Plasmid) maintains the Fosmids at a single copy in the cell.

BACs can carry even larger DNA pieces than Cosmids

Conjugative F-plasmid can integrate into the bacterial chromosome

BACs use F-plasmid elements

Cloning Vectors for Eukaryotes

The yeast 2 μm plasmid

The development of cloning vectors for yeast was initially stimulated by the discovery of a plasmid that is present in most strais of S.cerevisiae. The 2µm plasmid is one of only a very limited number of plasmids found in eukaryotic cells.

The yeast 2 μm plasmid is a selfish DNA element.

2 μm plasmid can ‘flip’!

FLP = Flippase Recombinase 

FRT = Flippase Recognition Target


Flipping‘ corrects decreases in 2 µ plasmid copy number. It does so by causing recombination between the two inverted repetitions (FRTs) during DNA replication. This changes the direction of one replication fork, causing multiple rounds of copying in a single initiation.

FLP / FRT is being used for genetic engineering in different organisms (e.g. in flies to turn on/off a gene of interest (GOI)

Selectable markers for the 2 !m plasmid

  1. in order to use LEU2 as a selectable marker, a special kind of host organism is needed. The host must be an auxothrophic mutant that has a non-functional LEU2 gene. Such a leu2-  yeast is unable to synthesize leucine and can survive only if this amino acid is supplied as a nutrient in the growth medium.
  2. Selection is possible because transformants contain a plasmid-borne copy of the LEU2 gene, and so are able to grwo in the absence of the amino acid. In a cloning experiment, cells are plated out onto minimal medium, which contains no added amino acids. only transformed cells are able o survive and form colonies.

Cloning vectors for mammalian cells

Purpose

Gene knockout: to study the function of genes - e.g. CRE / FLP recombinations

Pharming: production of recombinant protein in mammalian cell culture, - e.g genetic engineering of a farm animal so that it synthesizes an important protein such as pharmaceutical, often in its milk

Gene therapy: engineering human cells to treat a disease

pCDNA3.1 allows constitutive expression of the cloned DNA in mammalian cells via the CMV promoter

The CMV promoter is commonly included in vectors used in genetic engineering work conducted in mammalian cells, as it is a strong promoter and drives the constitutive expression of genes under its control.

Human Cytomegalovirus (HCMV)

1. Binding of HCMV glycoproteins gB and gH to cellular receptors activates cellular transcription factors (NF-κB and Sp1).

2. Virus enters the cell, releases viral DNA, proteins, and mRNA transcripts into the cytoplasm.

3. Viral mRNAs are translated, viral DNA, and certain viral proteins are transported to the nucleus.

4. In the nucleus, viral genes are expressed, with help from the activated transcription factors, and viral DNA is replicated.

5. Viral DNA, RNA, and proteins are packaged into the virion and infectious viral particle is released.


belongs to Herpes Viruses. enveloped with icosahedral structure. Genome is linear ds DNA. largest genome of any known human virus: 236 kbp! infected bodies retain CMV for life. most people don't know they have CMV because it rarely causes problems in healthy people.


Lipofection = Lipid Transfection = liposome-based transfection


(A) The cell membrane is composed of a lipid bilayer, with a hydrophobic interior and hydrophilic exterior. 

(B) Liposomes are also composed of a lipid bilayer arranged as a spherical shell. 

(C) A brief incubation of lipid-based reagents allows liposomes to form around DNA. 

(D) Cells in culture can endocytose the liposome, digesting it within vesicles to release DNA. 

(E) Alternatively, liposomes can directly fuse with the plasma membrane, directly releasing DNA into cells.

LipofectamineTM is a highly used Transfection Reagent

Viral vectors

Retroviruses, Adenoviruses, and Adeno-associated virus (AAV)

Retroviruses, such as Lentiviruses are commonly used vectors for gene therapy. Although they insert at random positions, the resulting integrants are very stable, which means that the therapeutic effects of the cloned gene will persist for some time. The best-known lentivirus is the human immunodeficiency virus (HIV), which causes AIDS.

Adenoviruses, which enable DNA fragments of up to 8 kb to be cloned, longer than is possible with an SV40 vector, though adenoviruses are more difficult to handle because their genomes are bigger.

Adeno-associated virus (AAV), which is unrelated to adenovirus but often found in the same infected tissues, mainly because AAV utilizes some of the proteins synthesized by adenovirus in order to complete its replication cycle. In the absence of this helper virus, the AAV genome inserts into its host’s DNA. With most integrative viruses this is a random event, but AAV has the unusual property of always inserting at the same position, within human chromosome 19. Knowing exactly where the cloned gene will be in the host genome is important if the outcome of the cloning experiment must be checked rigorously, as is the case for applications such as gene therapy. AAV vectors are therefore considered to have major potential in this area.

The early cloning experiment with mammalian cells was in 1979 with a vector-based on Simian Virus 40 (SV40).

(a) SV40 and (b) an example of its use as a cloning vector. To clone the rabbit β-globin gene, the HindIII to BamHI restriction fragment was deleted (resulting in SVGT-5) and replaced with the rabbit gene.

SV40 suffers from the same problem as λ: packaging constraints limit the amount of new DNA that can be inserted into the genome.

Cloning with SV40 therefore involves replacing one or more of the existing genes with the DNA to be cloned.

a) This virus is capable of infecting several mammalian species, following a lytic cycle in some hosts and a lysogenic cycle in others. The genome is 5.2kb in size. It contains two sets of genes, the éarly´genes, which are expressed early in the infection cycle and code for proteins involved in viral DNA replication, and the ´late´genes, which code for the viral capsid proteins. 

Adenoviruses


Adenoviruses have a unique ability to infect a broad range of cell types. adenovirus-based vectors can be used to transduce and deliver transgenes to different cell types. adenovirus vectors do not integrate into host genomes but stay as episomal DNA in the nucleus of host cells. major drawbacks: induction of undesired innate immune responses. - in liver and spleen, the resident macrophages can sense and trap blood-borne adenovirus and induce inflammatory response mediators. immunogenicity, cellular toxicity, and oncogenesis are also major obstacles in gene therapy applications

Retroviruses include Lentiviruses such as the human immunodeficiency virus (HIV)

a retrovirus inserts a copy of its RNA genome into the DNA of a host cell. the virus uses its own reverse transcriptase to produce DNA from its RNA genome (‚retro‘). incorporated retroviral DNA is referred to as a provirus. host cell treats the viral DNA as part of its own genome. 

>> transcribing and translating the viral genes.

>> producing viral proteins to assemble new virus particles.

General Retrovirus cycle

Organization of the Retrovirus genome.

The LTR (long terminal repeat) consists of replication-initiation sequences, transcription- and translation-regulatory sequences (U3 and U5), as well as sequences necessary for transgene integration (R: repeated sequences).

Retrovirus genome stages

Transient production of lentivirus vectors based on three-plasmids transfection:

Lentivirus (Retrovirus) production

Adeno-Associated Virus (AAV)

AAV production need helper viruses

Different methods for capsid discovery and engineering to improve Tropism

Viral Tropism is the ability of different viral strains to infect different cell types or tissues.

Recombinant AAVs (rAAVs): single-stranded AAV (ssAAV) and self-complementary AAV (scAAV)

Timeline of AAV gene therapy development

Gene Therapy for Cystic Fibrosis (AAV first applied in 1995)

2012: first marketing authorization for gene therapy in either EU or US

Glybera, is a gene therapy treatment designed to reverse lipoprotein lipase deficiency (LPLD), a rare inherited disorder which can cause severe pancreatitis. It was approved by the European Commission in 2012 and thereby the first marketing authorization for a gene therapy treatment in either Europe or the United States.

Lipoprotein lipase deficiency (LPLD)

Lipoprotein lipase deficiency (LPLD) is a genetic disorder. A defective gene for lipoprotein lipase, which leads to very high triglycerides, causes stomach pain and deposits of fat under the skin. Also, it can lead to problems with the pancreas and liver, which in turn can lead to diabetes. The disorder occurs if a child acquires the defective gene from both parents (it is autosomal recessive). Without gene therapy it is managed by restricting fat in diet to less than 20 g/day.

Treatment of LPLD with AVV for gene therapy (first approved!)

Glybera The adeno-associated virus serotype 1 (AAV1) viral vector delivers an intact copy of the human lipoprotein lipase (LPL) gene to muscle cells. The LPL gene is not inserted into the cell's chromosomes but remains as free floating DNA in the nucleus.

Retinal Dystrophy (loss of vision): Retinitis pigmentosa (RP)

2017, the FDA approved (Luxturna) as a gene therapy treatment for individuals with inherited retinal diseases such as retinitis pigmentosa. For this treatment, an AAV2 vector carrying the gene RPE65 is delivered by injection into the subretinal space.

AAVs to produce therapeutic proteins in the liver

Somatic cell therapy with stem cells in vitro

Differentiation of a transfected stem cell leads to the new gene being present in all the mature blood cells.

Overview of rAAV interventional gene therapy clinical trials

AAV gene therapy literature (recommended to read during the break)

Cloning Vectors for Eukaryotes

Lecture 10.12

Principle of a Recombination System

Cre-Lox recombination System is derived from the P1 Phage

Cre Recombinase enzyme and the LoxP sequence are derived from bacteriophage P1

During lysogenic cycle the phage genome exists as a plasmid in the bacterium unlike lambda, which integrates into the host DNA.

Cre-Lox recombination System allows gene activation/inactivation in transgenic cells

Cre-Lox recombination System allows gene activation / inactivation in transgenic animals

FLP / FRT is being used for genetic engineering in different organisms (e.g. in mice to turn on/off a gene of interest (GOI)

Yeast Cloning Vectors (often based on 2 μm plasmid

Episomal plasmids: Yep = Yeast Episomal plasmid

Vectors derived from the 2µm plasmid are called yeast episomal plasmids (YEps). Some YEps contain the entire 2µm plasmid while others include just the 2µm origin of replication. An example of the latter type is YEp13.

Cloning with an E. coli–yeast shuttle vector such as YEp13.

The standard procedure when cloning in yeast is to perform the initial cloning  experiment with E.coli, and to select recombinants in this organism. Recombinant plasmids can then be purified, characterized, and the correct molecule introduced into yeast.

Recombination between plasmid and chromosomal LEU2 genes can integrate YEp13 into yeast chromosomal DNA

The word "episomal" indicates that a YEp can replicate as an independent plasmid, but also implies that the integration into one of the yeast chromosomes can occur. Integration occurs because the gene carried on the vector as a selectable marker is very similar to the mutant version of the gene present in the yeast chromosomal DNA. With YEp13 homologous recombination can occur between the plasmid LEU2 gene and the yeast mutant LEU2 gene, resulting in insertion of the entire plasmid into one of the yeast chromosomes. The plasmid may remain integrated, or a later recombination event may result in it being excised again.

Other types of cloning vector for use with S. cerevisiae

  1. Yeast integrative plasmids (YIps) are basically bacterial plasmids carrying a yeast gene. An example is YIp5, which is pBR322 with an inserted URA3 gene. This gene codes for orotidine-5´-phosphate decarboxylase and is used as a selectable marker in exactly the same way as LEU2. A YIp cannot replicate independently as it does not contain any parts of the 2µm plasmid, and instead depends for its survival on integration in yeast chromosomal DNA.
  2. Yeast replicate plasmids (YRps) are able to multiply as independent plasmids because they carry a chromosomal DNA sequence that includes an origin of replication. Replication origins are knwon to be located very close to several yeast genes, including one or two which can be used as selectable markers. 

YEp, YRp, or YIp

Transformation frequency 

YEps: 10 000 and 100 000 transformed cells per microgram. 

YRps: 1000 and 10 000 transformants per microgram, 

YIp: less than 1000 transformants per microgram, (chromosomal integration event is necessary before the vector can be retained in a yeast cell)

Copy number 

YEps and YRps: 20–50 and 5–100 copies per cell 

YIp: one copy per cell.

If the objective is to obtain protein from the cloned gene >> more copies >> more yield of the protein product >> use YEps or YRPs.

Why Ylps? 

YIps produce very stable recombinants, as the loss of a YIp that has become integrated into a chromosome occurs at only a very low frequency. YRp and YEp recombinants are extremely unstable, daughter cells often non-recombinant.

Artificial chromosomes require ‘natural’ features

Eukaryotic Chromosomes usually need multiple replication origins

Yeast artificial chromosome (YAC) works also with one replication origin



pYAC3 is essentially a pBR322 plasmid into which a number of yeast genes have been inserted. The DNA fragment that carries TRP1 also contains an origin of replication, it also includes CEN4, which is the DNA from the centromere region of chromosome 4. SUP4 is the selectable marker into which new DNA is inserted during the cloning experiment.


disruption of SUP4 gene causes ‘white’ yeast colonies. functional SUP4 gene makes yeast colonies turn red.

SUP4 gene allows selection for recombinants

disruption of SUP4 gene causes ‘white’ yeast colonies.

functional SUP4 gene makes yeast colonies turn red.

Yeast artificial chromosome (YAC)


The vector is first restricted with a combination of BamHI and SnaBI, cutting the molecule into 3 fragments. The fragment flanked by BamHI sites is discarded, leaving two arms, each bounded by one TEL sequence and one SnaBI site. The DNA to be cloned, which must have blunt ends, is ligated between the two arms, producing the artificial chromosome.



Purpose for YACs

Several important mammalian genes are greater than 100 kb in length (e.g., the human cystic fibrosis gene is 250 kb), which is beyond the capacity of all but the most sophisticated E. coli cloning systems. YAC vectors are routinely used to clone 600 kb fragments, and special types are able to handle DNA up to 1400 kb in length, the latter bringing the size of a human gene library down to just 6500 clones. Unfortunately, these ‘mega-YACs’ have run into problems of insert stability, the cloned DNA sometimes becoming rearranged by intramolecular recombination. BACs turned out to be more stable (single copy!).

Yeast applications: identify networks of protein interactions.

Yeast two-hybrid analysis reveals networks of protein interactions.

Yeast two-hybrid: a selection gene is only activated upon interaction of tested proteins

Synthesis of a quadrivalent HPV vaccine. The four recombinant yeast strains synthesize L1 proteins from HPV subtypes 6, 11, 16, and 18.


Recombinant vaccines have also been successfully developed for human papillomavirus (HPV), different subtypes of which are responsible for a variety of cancers. The L1 coat protein gene has been expressed in S.cerevisiae, giving rise to virus-like particles composed of aggregates of the L1 protein. These particles lack any nucleic acid and so are not infectious. In order to broaden the effectiveness of recombinant HPV vaccines, a variety of recombinant yeast strains have been produced, each synthesizing the L1 protein from a different HPV subtype. The virus-like particles from the strains are then combined to make a divalent or quadrivalent vaccine.

Cloning vectors for plants

Agrobacterium tumefaciens: nature’s smallest genetic engineer

A. tumefaciens genetically engineers the plant cell for its own purposes.

Agrobacterium tumefaciens: causes ‘crown gall’ disease


The Ti plasmid of Agrobacterium tumerfaciens is of great importance. Crown gall occurs when a wound on the stem allows A.tumerfaciens bacteria to invade the plant. After infection, the bacteria use a cancerous proliferation of the stem tissue in the region of the crown.

The Ti plasmid integrates into the plant chromosomal DNA after A. tumefaciens infection


The Ti plasmid is large (>200kb). It carries numerous genes involved in the infective process. A remarkable feature of the Ti plasmid is that, after infection, part of the molecule is integrated into the plant chromosomal DNA. The T-DNA is between 15 and 30b in size, depending on the strain. The T-DNA is maintained in a stable form in the plant cell and is passed on to daughter cells as an integral part of the chromosomes. The most remarkable feature of the Ti plasmid is that the T-DNA contains eight or so genes that are expressed in the plant cell and are responsible for the cancerous properties of the transformed cells. These genes also direct the synthesis of unusual compounds, called opines, that the bacteria use as nutrients.

T-DNA contains around eight genes that are expressed in the plant cell and are responsible for the cancerous properties of the transformed cell.


Using the Ti plasmid to introduce new genes into a plant cell


The binary vector strategy is based on the observation that the T-DNA does not need to be physically attached to the rest of the Ti plasmid.  Plasmid A and B complement each other when present together in the same A.tumerfaciens cell. The T-DNA carried by plasmid B is transferred to the plant chromosomal DNA by proteins coded by genes carried by plasmid

The co-integration strategy uses an entirely ew plasmid, based on an E.coli vector, but carrying a small portion of the T-DNA. The homology between the new molecule and the Ti plasmid means that if both are present in the same A.tumerfaciens cell, recombination can integrate the E.coli plasmid into the T-DNA region.

Transformation of plant cells by recombinant A. tumefaciens.

The binary Ti vector pBIN19 is ‘disarmed’

parts of the T-DNA that are involved in infection are two 25 bp repeat sequences found at the left and right borders of the region integrated into the plant DNA.

Genetically modified (GM) crops

Genetically engineered rice containing a biosynthetic pathway for β-carotene.

Glyphosate (ROUNDUP®) Resistance in Plants

Glyphosate Resistance in Plants

Lecture 7.1.2021

Transgenesis in Animals example: C. elegans

DNA is injected directly into the germline of ‘mothers’

Selection issue: what if recombinant DNA or Editing is ‘invisible’?

Co-injection with a visible recombinant DNA… fluorescence…

Tissue-specific and inducible expression

Transgenesis in Mice

pCDNA3.1 allows constitutive expression of the cloned DNA in mammalian cells via the CMV promoter.

The CMV promoter is commonly included in vectors used in genetic engineering work conducted in mammalian cells, as it is a strong promoter and drives the constitutive expression of genes under its control.

Cre-Lox recombination to inactivate genes in specific tissues

Tetracycline-controlled transcriptional activation

Tet-On 3G activates transcription upon Doxycycline binding

Tet-On 3G protein: fusion of the repressor (TetR), with the activation domain of another protein, VP16, found in the Herpes Simplex Virus.

P = Promoter

‚TRE‘ contains 7 repeats of a 19 nucleotide sequence bound by the tetracycline repressor (TetR)

Generating tetracycline-inducible gene constructs

Using the Tet-On system in transgenic mice

In contrast: ‘somatic’ gene therapies (can not be inherited)

2017, the FDA approved (Luxturna) as a gene therapy treatment for individuals with inherited retinal diseases such as retinitis pigmentosa. For this treatment, an AAV2 vector carrying the gene RPE65 is delivered by injection into the subretinal space.

Stem cell method

Delivery: Lipofection = Lipid Transfection = liposome-based transfection


(A) The cell membrane is composed of a lipid bilayer, with a hydrophobic interior and hydrophilic exterior.

(B) Liposomes are also composed of a lipid bilayer arranged as a spherical shell.

(C) A brief incubation of lipid-based reagents allows liposomes to form around DNA.

(D) Cells in culture can endocytose the liposome, digesting it within vesicles to release DNA.

(E) Alternatively, liposomes can directly fuse with the plasma membrane, directly releasing DNA into cells.


Viral Vectors

Delivery: Microinjection

Neomycin: an antibiotic that is also toxic for mammalian cells!

Neomycin is an aminoglycoside antibiotic found in many topical medications such as creams, ointments, and eyedrops.

Mammalian cells will eventually die off when the culture is treated with high doses of neomycin or a similar antibiotic.

Geneticin (G418) is a neomycin analog and toxic for eukaryotes. G418 blocks protein translation in both prokaryotic and eukaryotic cells.

Neomycin and G418 resistance are conferred by aminoglycoside phosphotransferase genes. A neoR gene is commonly included in DNA plasmids to establish stable mammalian cell lines expressing cloned proteins.

Neomycin selection

for mammalian cells concentrations of approximately 400 μg/mL, G418 is used.

Neomycin / G418 selection: determine the optimal concentration for each cell line and batch!!


Also note: some cell lines are already resistant! e.g. the Human Embryonic Kidney 293 cells ‘HEK293’ are not, but HEK293T can not be killed by neomycin.


The antibiotic ‘Puromycin’ can also be used for the selection

Puromycin is an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger.

It is a potent translational inhibitor in both prokaryotic and eukaryotic cells.

Resistance to puromycin is conferred by the puromycin N-acetyl-transferase gene (pac).

Puromycin has a fast mode of action, causing rapid cell death at low antibiotic concentrations.

Mammalian cells are sensitive to concentrations of 0.5 to 5 µg/ml.

Puromycin-resistant stable mammalian cell lines can be generated in less than one week.

Homologous Recombination: site-specific recombination

Site-specific homologous recombination for genomic engineering

Site-specific homologous recombination for genomic engineering

Selection for recombination event: e.g. Neomycin

Homologous Recombination occurs naturally; e.g. during Meiosis by ‘Crossing-Over’

Homologous Recombination requires DNA breaks

viruses that can integrate into the host genome like Retroviruses and AAV bring along their own DNA endonucleases!


inverted terminal repeat (ITR) of AAV consists of two small palindromes.

rep proteins are required for integration into host genome >> have endonuclease activity!



Need to induce DNA breaks for homologous recombination! The restriction endonuclease Fok1 – attached to DNA-binding protein sequences: Zinc Finger

Inducing DNA breaks for homologous recombination: Fok1 – attached to polymorphic amino acid repeats


TAL = transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. 

EN = Endonuclease (Fok1)


Class 2 Clustered Regularly Interspaced Short Palindromic Repeat revolution

CRISPR/Cas9 is the latest technology for the generation of genetically modified animals.

CRISPR/Cas9 technology for genetic modifications in animals

Flowchart of different methodologies for mouse transgenesis


Neomycin selection

for mammalian cell concentrations of approximately 400 μg/mL, G418 is used.

Stem cell method

Site-specific homologous recombination for genomic engineering

Site-specific homologous recombination for genomic engineering

Selection for recombination event: e.g. Neomycin

DNA nucleases induce DNA Double-Strand Breaks (DSBs), which are important for genetic engineering to generate GMOs


CRISPR/Cas9 is a natural RNA-mediated bacterial adaptive immune system

CRISPR discovery timeline

Natural CRISPR/Cas9 system (Type II)

CRISPR repeat clusters are separated by non-repeating DNA sequences called spacers, which represent sequences of viruses and other invaders.

Cas9 has two catalytic domains (HNH and RuvC) that act together to mediate DNA Double-Strand Breaks (DSBs). Each of these catalytic domains cleaves one DNA strand, thereby resulting in DSBs proximal to the PAM sequence. A single point mutation in either of these domains results in a nickase enzyme = cuts only one strand! Mutations in both domains result in complete loss of DNA cleavage activity = dead Cas9 (dCas9)!

CRISPR/Cas9 principle

CRISPR/Cas9 induces DSBs

Major components of the CRISPR/Cas9 system

Cas9 = CRISPR associated protein 9 is an RNA-guided DNA endonuclease

CRISPR = Clustered Regularly Interspaced Short Palindromic Repeats

Derived from the Type II adaptive immunity system of Streptococcus bacteria

The tracrRNA is complementary to the CRISPR repeats.

The crRNA is complementary to the target sequence and contains a region that can bind to the tracrRNA.

The tracrRNA and crRNA form an RNA duplex which is recognized by Cas9.

Examples when targeting with one sgRNA in C. elegans (no ‘repair template’ for homologous recombination used!)

Glossary I of CRISPR/Cas9 system

Component
Function
cRNA
Contains the guide RNA that locates the correct segment of host DNA along with a region that binds to tracrRNA(generally in a hairpin loop fomr), forming an active complex.
tracrRNA
Binds to crRNA and forms an active complex.
sgRNA
Single-guide RNAs are combined RNA consisting of a tracrRNA and at least one crRNA
Cas9
An enzyme whose active form is able to modify DNA. Many variants exist with different functions: single-strand nicking, double-strand breaking, only DNA binding.
Repair template
DNA molecule used as a template in the host cell´s DNA repair process, allowing insertion of a specific DNA sequence into the host segment broken by Cas9.

The protospacer adjacent motif (PAM)

The protospacer adjacent motif (PAM) is a short sequence in the target DNA, immediately downstream of the gRNAmatching (target) sequence. But PAM is NOT part of the sgRNA! NGG sequence stretch can be found every 8 bp in the human genome.

The protospacer adjacent motif (PAM)

The canonical PAM is an NGG trinucleotide sequence, i.e., any nucleotide followed by two guanines (G). While the identity of the N is flexible, studies have reported that cytosines (C) are favored and thymines (T) are disfavored in this position. Specific interactions between Cas9 and the PAM are required for binding of target DNA and local strand separation prior to cleavage. Even a single mismatch in the PAM results in at least 74% reduced Cas9 activity, so gRNAs should always be designed to target sequences next to an NGG PAM. The PAM sequence is NOT PART OF THE sgRNA!!

The need for more PAM sequences

Cas9 Species/Variants and PAM Sequences

Cas9 has two catalytic domains (HNH and RuvC)

Cas12 was initially identified as Cpf1: a Cas9 Homolog with different DNA cutting property!

Cas12 (Cpf1) naturally requires a single sgRNA.

Cas12 (Cfp1) cuts DNA at target sites 3ʹ downstream of the PAM sequence in a staggering fashion, generating a 5ʹ overhang rather than producing blunt ends like Cas9.

An example for preparing a CRISPR/Cas9 experiment (part I)

A specific example for the annealing setup of the 2-part system gRNAs.

Preparing a CRISPR/Cas9 experiment (part II)

Cas9 can have off-target effects if sgRNAs are not designed properly!

Given that CRISPR systems have evolved as a defense system against viruses that tend to frequently mutate, a slightly less specific CRISPR system is advantageous to bacteria. But for genetic engineering, this is a huge problem!

DNA/RNA ‘Bulge Mismatch: One reason for CRISPR/Cas9 off-target effects

NGS to examine CRISPR/Cas9 off-target effects

Ways of avoiding off-target effects

Cas9 has two catalytic domains (HNH and RuvC) that act together to mediate DNA Double Strand Breaks (DSBs). Each of these catalytic domains cleaves one DNA strand, thereby resulting in DSBs proximal to the PAM sequence. A single point mutation in either of these domains results in a nickase enzyme. Mutations in both domains results in complete loss of DNA cleavage activity = dead Cas9 (dCas9)!

These approaches require two separate guide RNAs to be in a certain proximal distance >> probability of off-target effects is reduced.

CRISPR/Cas9 technology for genetic modifications in animals

Cas12 guidance needs only a specific crRNA which does not require a tracrRNA

Cas9 can have off-target effects if sgRNAs are not designed properly!

Given that CRISPR systems have evolved as a defense system against viruses that tend to frequently mutate, a slightly less specific CRISPR system is advantageous to bacteria.

But for genetic engineering, this is a huge problem!

CRISPR-Cas system can be used to recruit different (d)Cas-fusions

Major application areas of CRISPR-Cas-based technologies beyond genome editing

catalytically inactive dead Cas9 (dCas9): cannot cleave DNA but can still be guided to the target sequence.

dCas9 fusion to the deaminase APOBEC1 for single-base editing

Major strategies to recruit DNA- and chromatin-targeting and modifying enzymes via the CRISPR-Cas system

Additional natural CRISPR-Cas enzymes

RNA-targeting tools based on modified CRISPR/Cas systems

CRISPR/Cas9 technology to target liver diseases.

CRISPR/Cas9 technology for gene therapies

Plant Virus Resistance Through CRISPR/Cas9 Technology

CRISPR/Cas9-based gene drive: dominant inheritance

CRISPR/Cas9-based gene drive technology

CRISPR GLOSSARY II

CRISPR/Cas can be delivered using a plasmid expressing Cas9 and the sgRNA

CRISPR/Cas can be delivered using plasmids to express Cas9 and the sgRNA

CRISPR/Cas delivery as Plasmids, mRNA, and RNPs for mammalian cells


Stem cell genes are not active in Fibroblasts

Genetic Engineering (GE) to express Yamanaka factors in fibroblasts


Barrier Genes restrict Reprogramming

Genetic Engineering to inactivate reprogramming barrier genes

Genetic Engineering to inactivate reprogramming barrier genes

RNA interference (RNAi) is a posttranscriptional gene regulation mechanism:

small interfering RNAs (siRNA) induce the sequence-specific degradation of homologous messenger RNA (mRNA).

Gene silencing by RNA interference (RNAi): small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs)

The micro RNA (miRNA) silencing pathway has similarities

as12 guidan

RNAi in C. elegans is simple: delivering dsRNA by feeding bacteria

Producing dsRNA in bacteria for C. elegans feeding to induce RNAi

siRNA delivery strategies into mammalian cells

One non-viral delivery alternative of si/miRNAs: PAMAM dendrimers – nanoparticles produced by a synthetic polymerization process

Exogenously introduced RNA is often quickly degraded in host cells by several enzymatic activities. Hydrophilic nature and negative charge of RNA causes difficulties in penetration through the cell membrane. To achieve successful therapeutic use of siRNAs, it is necessary to design transfection carriers with optimized cellular uptake, low cytotoxicity, and without immunological responses. The PAMAM nanocomplexes have low immunogenicity, protect nucleic acids against degradation, and help delivery into cells.


Steps in the development of miRNA therapeutics

miRNA therapeutics in clinical trials

Multimeric pri-miRNA mimics

Ectopic expression of the miRNA miR-302 induces reprogramming

Intronic expression of miRNA miR-302

The miR-302-expressing SpRNAi- RGFP vector is introduced into human somatic cells by liposomal transfection. The miR-302 cluster is co- expressed with the encoded RGFP gene transcripts and further processed into individual mature miR-302 molecules by RNA splicing enzymes.

Hereditary amyloidogenic transthyretin (ATTRv) amyloidosis: caused by point mutations in the gene that encodes transthyretin (TTR)

Transthyretin (TTR) is a transport protein in the serum that carries the thyroid hormone thyroxine and retinol-binding protein bound to retinol. This is how transthyretin gained its name: Transports Thyroxine and Retinol.

Sites of TTR production and sites affected by the disease:

a | Sites of amyloid (defective protein aggregates) deposition (dark pink and blue)

b | Sites of transthyretin production (red).

c | Organs affected (green outlines).

Hereditary amyloidogenic transthyretin (ATTRv) amyloidosis

Hereditary amyloidogenic transthyretin (ATTRv) amyloidosis is an autosomal dominant disease. Presents as a progressive peripheral neuropathy. Is caused by point mutations in the transthyretin (TTR) gene.

(ATTRv) amyloidosis: 1st approved RNAi drugs: Patisiran & Inotersen 

Two categories of mRNA constructs for mRNA vaccines

Requirements of the mRNA construct to express the gene for producing the immunizing protein efficiently.

Considerations for effectiveness of a directly injected mRNA vaccine.

mRNA vaccine developers: research focus, partners and therapeutic platforms

Clinical trials with mRNA vaccines against cancer

Major delivery methods for mRNA vaccines.


Genetic Engineering

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