Methods

Method Development

Purification of AAV particles

IMAC purification via Viral Brick: The Histidin Affinity Tag

Protein tagging via Histidine Tags is a widely used method for protein purification: Multiple histidine residues (most commonly: Six) are being fused tot he end of the targeting protein. The high binding affinity of Histidine towards metal is being exploited for the purification of proteins via the so called „Immobilized Metal Ion Affinity Chromatography“ (IMAC): Multiple histidine residues (most commonly: Six) are being fused to the end of the targeting protein. A cell extract containing the recombinant protein ist then applied to a collumn containing immobilized Ni2+-Ions. The His-tags covalently bind the Ni-Ions while other cellular proteins can be washed oft he collumn. The purified proteins can then be eluted with Imidazol, which displaces the histidine residues.(MC Smith et al. 1988), (Hoffmann and Roeder 1991) Since the aim behind engineering therapeutic AAV vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. Contamination by cellular proteins could cause toxic side effects or a strong immune response. (Koerber et al. 2007) have first inserted a His-tag into a surface-exposed loop at amino acid position 587 in the Cap protein and successfully purified recombinant virsuses using IMAC. For our Virus Construction Kit, we provide the His-tag motif in the ViralBrick standard, allowing an easy insertion into the 453 and/or 587 loop.

Specific Biotinylation via ViralBrick: The Biotinylation Acceptor Peptide

The BAP (Biotinylation Acceptor Peptide) that we included in our Virus Construction Kit is a 15 amino acid long peptide identified by Schatz J., 1993 in an library screening approach and published under the number #85. This peptide with the sequence 5' - GLNDIFEAQKIEWHE - 3' contains a central lysine that is specifically biotinylated by the prokaryotic enzyme biotin holenzyme synthetase, encoded in the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by contransfecting a plasmid with the BirA gene as described for the AAV in Arnold et al.; 2006 or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA). The purified BirA biotin ligase that was kindly provided by Avidity.

Experiences from under the hood: Cell Culture

Optimizing the transfection protocol

Fig.1 : Comparison of transduction efficiencies viral stocks created with different 2xHBS Buffers

The optimization of the lab intern standard protocols was one of our fields on investigation. Besides optimizing the handling of our different cell lines and working steps, the transfection protocol was examined. One of the most remarkable lessons after several transfection performed was the crucial handling of the AAV293 cells. Once over approximately 80 % confluence the cells are no longer competent for transfection. Another achievement in method development was the determination of the optimal plasmid amounts. The best results were performed using 3.3 µg per plasmid and therefore this parameter was modified in our standard protocol. After transfecting the AAV293 we were able to detect the Ca2+-DNA conglomerates in the medium. The toxic side effects of these conglomerates were also confirmed. Not only the medium had to be changed, but also washing with PBS was essential to keep the cells alive.

The most critical steps in transfection is the exact pH of the 2x Hepes buffered-saline (HBS) buffer, in which the C2+-DNA complexes precipitate. Our initial buffer had a pH of 7.112. To determine the best pH-value for transfection, transfections were performed using buffers with different pH, harvested the viral particles and used the flow cytometry to define the optimal pH. Transfection, harvesting and transducton were performed according to the modified standard protocol.

After confirming that the highest amount of viral particles was created with the pH 7.112 2xHBS we wanted to determine how valid the flow cytometry data is and created a standard derivation.

Flow Cytometry Analysis

Fig.2: Ten transductions with the same viral vector for determination the standard derivation in flow cytometry

After transduction with equal volumes of the created viral stock the flow cytometry was performed according to FACS protocol. As it can be seen there is only a small standard derivation of 6.19% in mVenus positive cells. By combining the results gained from the experiments described above, we were able to evaluate the standard protocol described in Material and Methods.

Imaging AAV: EM/AFM pictures

To visualise our custom AAV particles, we took transmission electron microscopy as well as atomic force microscopy pictures from our virus samples:

AFM

AFM picture: Phase

AFM picture: Height

EM

EM picture 1

EM picture 2

Particles appear too large in size, according to Chen et al. this phenomenon might be due to aggregating particles.

Resources

1. Chen H. Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation. Scanning. 29(5):238-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17828711.

ITR cloning

Test Digestion of our final ITR BioBricks

As a part of our modularization of the AAV vector plasmids, we needed to extract the sequences making up the ITRs at each end of the vector and clone them into an IGEM-compatible backbone. But due to the ITRs’ strong secondary structures, none of our PCR-based approaches worked. External companies weren’t able to synthesize or even sequence the ITRs. Taking advantage of NotI and PstI restriction sites flanking the ITRs, we worked out a complex cloning strategy that finally led to functional ITR motives in the rfc10 standard.

See also: Hannas' ITR Diary

Serum-free cell culture medium

Introduction

Serum-free mediums allow users to standardize their cell culture conditions. It contains no animal proteins or animal-origin constituents, e.g. FCS (fetal calf serum).
The AAV-293 cells are used for AAV-2 production and are usually grown in (among other chemicals, such as nutrients, antibiotics, growth factors) serum supplemented DMEM medium. Regarding Western Blots, size exclusion chromatographies and other (purification) methods, the undefined and also highly variable serum products can disturb or interfere with these methods. Therefore it is useful for many applications to grow AAV-293 cells in serum-free medium.
Because our long term goal for AAV vectors is application in human patients, we are also trying to develop new methods to produce pure, uncontaminated AAV particles. The use of FCS to supplement cell culture medium for AAV particle production is problematic because even smallest amounts of animal antigens in the administered drug could lead to a strong immune response in patients.

Testing serum free medium

Serum free medium was obtained from AAV-293 cells are not adapted to serum-free growth conditions so we had to accustom them to the new growth conditions step by step, starting with 25% serum-free medium, e.g. 15 ml DMEM (not serum-free) + 5 ml serum-free medium (FreeStyle™ 293 Expression Medium) for a T75 flask. Each step takes at least 1 passage. We raised the serum-free ratio to 100 % over 7 passages.
100% serum-free cells grow slower compared to the serum-supplemented ones and we had to check them regularly via microscopy because the medium contains no pH indicator.

Results

Even though cells grew slower and handling was more difficult due to a missing pH indicator, we successfully cultivated AAV-293 cells in serum-free medium. The cells were used for AAV production, and we produced similar amounts of virus particles compared to cells grown in FCS-supplemented medium. Production efficiency can’t be compared exactly because after seeding cells for transfection they don’t grow as fast as the AAV-293 in serum containing medium.

Established Methods

Cloning

Cloning

Polymerase Chain Reaction

The Polymerase Chain Reaction (PCR) is a technique to amplify specific DNA sequences delivered by a DNA template independent from a bacterial system. Especially designed primers encompass the desired sequence. These primers serve as docking regions for the polymerase which extends the newly synthesized DNA strand.

The DNA template strand is heat denaturated (98 °C, 1 minute) to produce single-stranded DNA. The next step requires the temperature to be lowered to a temperature at which forward and reverse primers are able to anneal to their complementary bases on the DNA template. This temperature is defined by the length and the GC content of the primers. With increasing temperature (72 °C) the polymerase binds the priming regions and elongates the primers. The temperature is raised again to denaturate the double strand and the cycle starts anew.

PCRs were performed using Mastercycler gradient (Eppendorf, Hamburg, Germany), Mastercycler personal (Eppendorf) and Px2 ThermoHybaid devices (Thermo Fisher, Waltham, MA, USA). PhusionTM Polymerase together with corresponding buffer and dNTP mix were obtained from New England Biolabs (New England Biolabs, Ipswich, MA, USA).

Link to NEB: http://www.neb.com

Site-Directed Mutagenesis

The Site-Directed Mutagenesis (SDM) is used to mutate a specific base inside the plasmids sequence (Hutchison et al. 1978). Therefore, forward and reverse primers, which prime at the same site, containing a mismatch at the specific base in terms of the original structure are required. This mismatch defines the new base through which the original one is replaced.

As with Polymerase Chain Reaction, the site-directed mutagenesis works by amplifying the desired construct. The DNA double-strand is heat-denaturated which allows primers to bind to the single-stranded sequences after lowering the temperature. Designing a mismatch within the primers sequence leads to replacement of the unwanted base in later cycles of denaturating, annealing and elongation strands. After SDM program is finished, digestion with DpnI is necessary to digest parental methylated and hemi-methylated plasmid strands which do not contain the desired base pair exchange. For SDM, QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) were used.

Link to Agilent: http://www.genomics.agilent.com

Ligation

scematic representation of the idempotent cloning principle

Through restriction enzymes digested plasmid fragments can be reassembled into a new vector by ligation (see: Digestion). Ligase (Lehman 1974) connects complementary overhangs of fragments originated from digestion. The 5´phosphoryl group is bound to the hydroxyl group of the 3´end and therefore connects the fragments via ligase. This reaction requires energy whose form depends on ligase used. T4 DNA Ligase for example requires ATP. This new vector now holds the genetic information of both the opened vector (minus the cut out fragment), and the insert.

Transformation

Transformation is the process in which competent bacterial cells incorporate plasmid-DNA. DNA obtained from previous steps is added to competent cells. During incubation on ice, the plasmids attach to the cell surface. To make the cells assimilate the plasmids, the tubes are heat-shocked for a short time to 42 °C to allow the plasmids to pass the cell membrane. Although the mechanism is still not fully understood, it is probably related to a decrease in the cell’s membrane fluidity (Panja et al. 2008). After incubation on ice in order to regenerate the cells LB or DYT media is added and the tubes are incubated on a shaker at 37 °C to establish antibiotic resistance. After that, the cells are pelleted via centrifuging, the supernatant is discarded, and the pellet resuspended in the remaining rest of the media and plated on an agar plate containing the appropriate antibiotic. For transformation, BL21, XL1-blue and XL-10 Gold cells were used.

PCR purification

PCR purification is performed to purify the DNA sample from primers, salts, nucleotides, enzymes or other contaminations. This is based on different binding properties of these impurities on the membrane which is used within the purification. The PCR product together with a specific buffer which allows binding to the membrane is added on a column, which is centrifuged and the flow-through is discarded. Elution of the the PCR product is performed after washing the column several times For PCR purification, QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) was used. Used protocol: QIAquick ® (QIAGEN, Hilden, Germany)

Hybridisation of Oligos

Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. The main technique of a hybridization is that complementary strands of nucleic acids anneale after a heating and cooling down procedure. Denaturation of the double-stranded DNA unwinds it and separates it into single strands through the breaking of hydrogen bondings between the bases. In a renaturation step, the single strands finally hybridize and build double helices. Hybridisations can be performed using a Thermoblock or by running a PCR like we did in our experiments.

Fill-in-reactions

Scematic depiction of a Fill-in reaction using Klenow fragment

Fill-in reactions can be performed by PCR in order to blunt DNA. In comparison to a conventional PCR, the product is not obtained by amplification, but by fill-in 5'-overhangs of ssDNA using the Klenow-fragment [Figure]. This protein fragment is a product of the DNA polymerase I from E. coli, when cleaving it enzymatically by the protease subtilisin. It retains a 5' → 3' polymerase activity and a 5'→ 3' exonuclease activity, but is lacking the 3'→5' exonuclease activity. Therefore, the protein fragment is useful for many applications like DNA labeling by fill-in 5’-overhangs or strand displacement amplification (in this method the exo- klenow-fragment extends the 3'-end of a nicked strand and displaces the downstream DNA strand). After starting the PCR program, the samples are heated for 15 minutes at 94°C following an incubation time of 3 minutes at 94 °C. As soon as the samples are cooled down at 37 °C 1 µl Klenow-fragment (NEB, Frankfurt am Main) is added. The reaction is carried out usually in the same buffer as used for the digestion. While incubating the samples at 37°C for one hour, the fill-in reaction is finally running.

DNA gel electrophoresis

GeneRuler DNA ladder mix provided by Fermentas

Agarose gel electrophoresis and polyacrylamide gel electrophoresis are common analytical techniques to identify, quantify and purify nucleic acids. The usage of the two types of gels depends on the size of the fragments. Whereas an agarose gel is used to separate relatively long DNA molecules from 100 kDa up to 500 kDa, a polyacrylamide gel can also be used molecules shorter than 100 kDa. For our experiments we separated the DNA fragments by 1% - 1,5 % Agarose gel electrophoresis (Thermo EC Classic Series, Thermo Scientific) using GelredTM (Hayward, USA) for staining. GelredTM is a fluorescent nucleic acid gel stain which intercalates with nucleic acid and replaces the usage of the highly toxic ethidium bromide (EtBr) bit by bit. http://www.biotium.com

As a DNA ladder we used the GeneRulerTM DNA Ladder Mix (Fermentas, Germany).

Vector Dephosphorylation

A higher amount of vector backbone in cloning strategies can be reached by using a Phosphatase like the Antarctic Phosphatase (NEB, Frankfurt am Main, Germany). This enzyme catalyzes the removal of 5´ phosphate groups from DNA, RNA, rNTPs and dNTPs. [http://www.neb.com]. Dephosphorylated fragments cannot religate due to the lack of 5´ phosphoryl termini which are required by ligases. The dephosphorylation needs to be performed after digestion of the vector and before separating the fragments via agarose gel.

Gel Extraction

This technique is used to extract DNA fragments gained of previous cloning advances of 70 bp up to 10 kb after they have been separated by gel electrophoresis. The fragments of interest are identified by theoretically cloning of vector and/or insert with Geneious to get their fragment sizes and comparing them with a standard DNA ladder. The expected bands are visualized under UV light and isolated from the gel by using a scalpel (cleaned with EtOH before). Usually, the DNA is then extracted via a spin column extraction kit to remove the accompanying salts and stain. In our case, we used the QIAquick Gel extraction Kit (250) (QIAGEN, Germany) and the including protocol. The kit works on a silica-membrane basis and makes it possible to purify up to 400 mg slices of DNA bands from gels. After several fast binding and washing procedures the DNA is eluted with 30–50 μl elution buffer. Besides the PCR products, which were mostly eluted with 30 µl elution buffer, we usually used 20 µl of elution buffer. The purified DNA fragments are ready for direct use in all applications, including sequencing, ligation and transformation (QIAGEN, Hilden, Germany).

Verification of Correct BioBrick Part Assembly

After production of a new BioBrick vector, there are different possibilities to test the correct assembly of the used BioBrick parts before sending them for sequencing.

Test digestion

It is important to be aware of negative controls for the analysis of a test digestion. That means the vector backbone of the particular construct has to be digested with the same enzymes as the assembled BioBrick for comparing the expected bands. The required enzyme volume per sample can be reduced down to 0,5 µl of each enzyme. According to that, an incubation time of 40 min is enough and depending on the size of the expected fragments, the gel runs only about 25 min at 115 V.

Colony PCR

Another strategy is that a colony PCR can be performed by using primers that anneal to the verification primer binding sites. When designing these primers, it is important to make several predictions, for example to avoid undesirable pairings of the primers or unspecific bindings to the template. Therefore the primers are designed by choosing the same melting temperature (Tm) as the desired template. According to that they should have a primer length of about 800 bp for a good detection on the gel. With a colony PCR, bacterial colonies are screened directly by PCR. Each colony is picked with a sterile toothpick, which can be transferred not only into a PCR mix, but at the same time into fresh DYT media preparing for a Mini- or Midiprep. When using a standard polymerase, the PCR is started with an extended denaturation time of 95°C to release the DNA from the cells. After running the program, the samples are loaded on an agarose gel to proof the success of a BioBrick assembly.

Purification of Plasmid DNA

Miniprep

We used the QIAprep® Spin Miniprep Kit (QIAGEN, Germany) which enables the purification of up to 20 µg molecular biology grade plasmid DNA or cosmid DNA. The procedure was done following the QIAGEN standard protocol including three basic steps. First one is the clearing of the bacterial lysate, then the adsorption onto a membrane and at the end the elution of plasmid DNA. Before clearing the lysate, the bacteria are lysed under alkaline conditions (Buffer 1 with RNase, Buffer P2). Buffer P2 contains SDS, which solubilizes the phospholipid and protein components of the cell membrane, while NaOH denatures the plasmid DNAs and proteins. After that the lysate is subsequently neutralized (N3 Buffer) and exposed to high salt-binding conditions. This leads to precipitation of chromosomal DNA, cellular debris and SDS, while the smaller plasmid DNA renaturates and stays in the solution. As a next step the sample can be applied to the spin column following two washing steps with PB and PE Buffer. Finally, the DNA can be eluted with water or with elution buffer. Unlike the standard protocol, we eluted the DNA with 60 µl Buffer EB instead of using 50 µl. (QIAGEN, Hilden, Germany)

Midiprep

The Midipreps were done following the standard protocol of Qiagen using a QIAGEN® Plasmid Plus Midi Kit (QIAGEN, Hilden, Germany). These kits enable fast, large-scale purification of up to 250 μg of highly pure plasmid DNA (description of most used buffer under 3.1.12.1). By using a vacuum manifold which replaces the single centrifugation steps up to 24 samples can be prepared in parallel. Therefore we used the vacuum technique for large sample numbers of Midipreps as well as Minipreps. The plasmid DNA obtained is suitable for example to transfect the DNA into sensitive cell lines. (QIAGEN, Hilden, Germany)

Protocols; Standard Operating Procedures

Standard Protocol: Cloning

Media:Freiburg10_Advanced_Cloning_Protocol_04_08_2010.pdf‎‎
Media:Split_cellculture.pdf
Media:Freeze_cellculture.pdf
Media:production of competent E.coli.pdf
Media:Freiburg10_Transfection_protocoll.pdf
Media:Freiburg10 Thawing cells.pdf
Media:Freiburg10_Aminoacids_vs_restrictionsites.pdf
Media:Freiburg10 Endotoxinfreie Midi.pdf
Media:Freiburg10_LB+Agar.pdf‎
Media:Freiburg10_Subcloning_cap_into_pAAV_RC.pdf‎
Media:Freiburg10_Quantitative_realtime_PCR_for_Titering_of_infectious_AAV_particles.pdf