Optimized Manufacture of Lentivirus Using FuGENE® HD Transfection Reagent

Lentiviral vectors are ideally suited vehicles for just a wide range of research applications. Based on pseudotype, lentiviruses infect a variety of cell types, non-discriminately transducing both dividing and non-dividing cells. As opposed to other popular vector delivery systems, lentivirus stably and rapidly integrates genetic payload in the host genome enabling long-term studies in vivo. Current lentiviral vector systems can accommodate upwards of ten kilobases of foreign DNA [1], although promoter and enhancer elements slow up the practical height and width of gene open-reading frames to perhaps 5-6 kilo­bases, which is sufficient to accommodate most genes commonly studied within the mammalian genome. Genetic integrants also adopt native chromatin conformation ideal for easy use in gene regulation studies [2] and particular lentiviral pseudotypes are actually exploited for selective infectivity whereby cells interesting are preferentially transduced; as an example, VSV-G pseudotype displays broad tropism but preferentially targets neurons within the nerve fibres. Aside from the ability to transduce virtually all immortal cell lines with high efficiency, lentivirus has been used successfully in the growth and development of transgenic animals and vivo types of disease (reviewed in [3]), and involvement in gene therapy applications is constantly on the increase.

Research laboratories can produce current-generation lentiviral vectors in approved biosafety level-two containment areas with the necessity for specialized equipment. For optimal safety consideration, the next generation of lentivirus affords the very best level of protection, whereby minimal genetic elements are split among four plasmids that need to be expressing simultaneously in individual cells for successful viral production [4-6]. After transfection and subsequent expression with the proteins encoded because of the transiently transfected plasmids, the cell medium supernatant contains active lentiviral particles immediately usable to transduce other cells of great interest. However, investigators routinely report problems in obtaining sufficient levels of lentivirus for particular projects, and lenti­virus used in vivo has experienced varied reports of success within the ­literature. Difficulties with low titer and toxicity in purified preparations plague common entry to lentiviral vectors. Traditional methods for manufacture of lentiviral titers utilize calcium phosphate precipitation to transfect some required plasmids in to a packaging cell line, mostly HEK-293T cells [7]. Within our experience, this standard approach leads to high variability in transfection efficiencies and corresponding titers, and also the concentrated virus can be highly toxic to cells in downstream experiments.

Here, we describe many advances inside the production of lentiviral vectors. There are numerous new releases now commercially accessible which have clear benefit over predecessor equivalents. Using these protocols and associated products, we have virtually eliminated toxicity regarding lentiviral preparations and routinely obtain titers inside mid to high 1010 transduction units (TU) per milliliter, and, more importantly, obtain consistency in production.

The third-generation lentivirus system employs several enhanced safety measures over the second-generation packaging approach. Three packaging plasmids allow expression in trans of proteins required to produce functional virus. The packaging plasmids pLP1, pLP2 and pLP/VSVG (Invitrogen) were modified to prevent stretches of homology within the plasmids to prevent recombination as well as contain optimized promoter and enhancer elements for expression in HEK-293FT cells. These packaging plasmids are compatible with a number of lentiviral expression plasmids. To the experiments described here, a lentiviral vector encoding eGFP (cFUGW), WPRE, and cPPT was utilized. Preference ought to be directed at lentiviral expression plasmids containing the cPPT and WPRE elements that considerably increase transduction efficiency, specifically in primary cells.

Packaging cell line

A serious limitation and supply of variability in virus production stems from variations inherent in commercially accessible serum products, where lot-to-lot variations of 10,000-fold are already described for a few growth factors. We sought to remove the employment of serum products during virus production and identified a commercially accessible cell line (HEK 293-FT cells, Invitrogen) amenable to serum-free media formulations. 293-FT cells are clonal derivatives on the fast-growing 293-F cell line variant and stably express the SV40 large T antigen driven on the pCMVSPORT6TAg.neo construct. Over-expression from the large T antigen allows enhanced episomal replication of packaging and lentiviral expression plasmids containing the SV-40 origin of replication and usually enhances lentiviral production through additional unknown mechanisms. 293-FT cells were switched from the manufacturer´s ­recommended medium with a low/no serum-compatible medium (Opti-MEM, Invitrogen) supplemented with 5% fetal-bovine serum (FBS) and 500 µg/ml G418 for growth and maintenance. Culture vessels are maintained at 37°C at 5% CO2 in 95% RH.

Lentivirus supernatant production

HEK-293FT cells maintained in Opti-MEM (with Glutamax) supplemented with 5% FBS were plated to approximate 50% confluence in Nalge Nunc Nunclon T-175 flasks coated with polylysine (flasks were helped by 0.1 mg/ml polylysine for 60 minutes and washed 3 x with mineral water immediately previous to use). Sixteen hours later, the medium was substituted for 20 ml of serum-free Opti-MEM supplemented with 25 µM chloroquine. 10.5 µg LP1, 7 µg LP2, 10.5 µg pVSV-G, and 9 µg of lentiviral vector were included with 2 ml of serum-free Opti-MEM. 100 µl of FuGENE® HD Transfection Reagent was added as well as the mixture was briefly vortexed and incubated at room temperature for quarter-hour. This mixture was added straight to the HEK-293FT cell layer while the flask was gently agitated to distribute the transfection complexes. Eight hours later, 10 µM sodium butyrate was added right to the flask medium; 1 day following the addition in the transfection complex, the medium was discarded and replaced with 20 ml of serum-free Opti-MEM without any supplementation. Another twenty four hours later, the medium was collected in a 50-ml tube and another 20 ml of Opti-MEM was included with the T-175 flasks. Medium supernatant was centrifuged at 3,000 x g for ten mins and decanted into another 50 ml tube and held at 4˚C. After 1 day, a second collection was included with the 1st collection and centrifuged again at 3,000 x g for 10-20 minutes. The supernatant was filtered by way of a 44-µm membrane. Virus is aliquoted and stored at -20˚C. Thus, each T-175 flask yields 40 ml of viral supernatant that will yield 1-5 x 107 infective particles per milliliter. The protocol can be adjusted to support larger preparations by appropriate scaling.

Virus concentration

For lentiviral applications over and above cell line use, concentration is generally important to achieve high transduction rates. Moreover, concentration is needed for titer estimation using p24 protein measurements. To concentrate the viral supernatant, 100 µl of Opti-prep density gradient medium (Sigma) is added to Beckman Ultraclear centrifuge tubes, and ~38 ml of supernatant is added per tube. The density gradient medium prevents harsh pelleting and resuspension steps that otherwise diminish titer. The supernatant is centrifuged using a swinging bucket rotor for three hours at 50,000 x g plus the top 37 ml are manually removed having a pipette. The end 1 ml boasts a 40 x viral preparation helpful for ex vivo applications that has a usual titer up to ~5 x 108 TU per ml.

For in vivo applications, 1-ml 40 x aliquots are combined into sterile microcentrifuge tubes and centrifuged for 18 hours at 20,000 x g at 4˚C. The supernatant is carefully removed along with the pellet is resuspended by gentle manual­ pipetting on the limits of solubility using pre-warmed serum-free Opti-MEM. Viral pellets may also be dissolved in pre-warmed PBS, although Opti-MEM contains small quantities of protein that act surfactants to further improve virus solubility. Virus is aliquoted into 5-µl fractions and stored at -80˚C, through an expected titer of ~5 x 1010 TU per ml.

Titer estimation

HEK-293FT cells or other cells of interest are plated with low confluency onto polylysine-coated 96-well plates with 100 µl serum-free Opti-MEM per well. The very next day, lentivirus preparation is included in the primary well (recommended 50 µl for 1 x supernatant, 10 µl for 40 x concentrate, and a couple of µl for high concentration virus) and volume is delivered to 200 µl with additional Opti-MEM. After mixing, 100 µl are taken out of the very first well and included in another well, mixed, and 100 µl are stripped away from that well, combined with the following etcetera, thereby accomplishing single:2 dilution number of virus. If your lentivirus encodes a fluorescent protein, a rightly containing an easily countable volume of fluorescent cells is identified: each infected cell represents a transducing unit. If the lentivirus isn’t going to encode a fluorescent protein, cells are fixed and immunofluorochemistry is used to distinguish transduced cells.

Results and Discussion

The primary measures in optimizing production of lentivirus involved rectifying steps of possible variation and excretion of components that can cause downstream toxicity. We identified a cell line (HEK-293FT cells) that’s amenable to low or no serum formulations that produce the best stages of lentivirus. Besides Opti-MEM, which contains animal-derived components, we used two other medium formulations which are chemically defined, namely CD-293 medium and Freestyle-293 media (Invitrogen). We experienced a 20% reduction of lentivirus production when substituting to CD-293 medium, and an 80% decrease in lentivirus production with Freestyle-293 medium (data not shown).

Traditional calcium phosphate transfection produces toxi­city in cells, creating cell-lifting problems, and necessitating additional medium changes. Cell lifting and death during viral production releases cell components that become co-concentrated with viral particles, thereby introducing toxic components to viral preparations. We found that FuGENE® HD Transfection Reagent co-transfects the 4 plasmids needed to produce virus with 100% efficiency into low-serum modified HEK-293FT cells. FuGENE® HD Transfection Reagent, as opposed to calcium phosphate, won’t cause cell lifting and death, plus doesn’t need an intermittent medium change.

4511-53We sought to help expand maximize viral production and transfection efficiency of cells transfected using FuGENE® HD Transfection Reagent through pre-management of cell lines with chloroquine and further treatment with sodium butyrate­ (Figure 1). Chloroquine is shown to lessen the degradation of plasmid-containing transfection complexes through partial neutralization on the pH within lysosomal compartments [8]. We found that chloroquine gives both immediate and long-term help to viral production, probably by helping the effective concentration of active plasmid DNA in cells. Sodium butyrate, which exerts a broad-spectrum effect on transcriptional activity, likewise increases viral production presumably through a combination of up-regulating viral promoters and boosting protein production output capability [9]. The amalgamation of chloroquine and sodium butyrate treatment modestly increases viral output compared with either treatment alone, perhaps caused by a maximal viral output limitation of transfected cells.

4512-53In vivo entry to lentivirus demands particularly pure and concentrated preparations. To determine whether or not the protocol described herein produced virus from the required caliber, we injected a couple-µl number of 4 x 1010 TU virus en­coding eGFP in to the anterior region of the mouse button caudate ­putamen (Figure 2). After 7 days, we analyzed brain sections through immunohistochemistry (Figure 2, panel a) and immunofluorescence (Figure 2, panel b). Broad infectivity with minimal toxicity throughout the needle site and with the striatum demonstrates the premium quality of ­lentiviral preparations produced using FuGENE® HD Transfection Reagent.

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Get rid of HIV and aggressive sort of leukemia ?

Scientists in the Laboratory of Molecular Virology and Gene Therapy of KU Leuven found that a protein through an important role inside the spread of HIV , exactly the same function exercises from the development of a hostile form of leukemia . They aspire to mitigate with the exact same inhibitor in the foreseeable future. Leukemia and HIV

Proteins inside cells with the your body shapes by their interactions a network that provides the ” control ” of the cell. To adopt above the ‘ governance’ on the cells will viruses , like HIV , efficiently manipulate certain network nodes to influence the network. A comparable phenomenon occurs while using growth of cancer . Again, make modifications in certain proteins to modifications in nodes on the network . Now it would appear that some nodes be involved both in the spread of HIV and the development of cancer .

It turned out shown which the protein LEDGF/p75 is an important hub for the multiplication of HIV . Genital herpes used this protein to anchor . Binds towards DNA from the cell Exactly the same protein plays an identical role inside the growth of certain forms of leukemia . For instance , an exceptionally aggressive sort of leukemia that always occurs in children younger than one full year. Meanwhile, have been developed which might be potent inhibitors from the interaction between this protein and HIV , so because of this and the DNA , the anchoring block of HIV.

Dr. Jan De Rijck , from the Laboratory of Molecular Virology and Gene Therapy led by Professor Zeger Debyser , showed now jointly researchers on the University of Basel that strategies that had been previously used also to inhibit this aggressive sort of leukemia cells in laboratory animals and HIV can stop . Inside a next thing , one will develop new drugs to manage leukemia , on such basis as these results.

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German Researchers Find Sensitizer for Gemcitabine Using DECIPHER RNAi Libraries

Researchers for the German Cancer Research facility (DKFZ) used Cellecta’s shRNA DECIPHER libraries to spot genes that sensitize pancreatic cells to gemcitabine.

Pancreatic cancer only has a 6% 5-year survival rate. Gemcitabine could be the standard treatment in partnership with surgery to take out cancerous pancreatic tissue from the minority with the cases when the ailment is caught in time. However, the gemcitabine response rate is below 20%, and combinations with targeted drugs are yet to improved this significantly until now.

To deal with this treatment problem, Dr. Michael Boettcher’s group at DKFZ used DECIPHER short-hairpin RNA (shRNA) libraries running loss-of-function genetic screens on pancreatic cancer cells to spot genes that enable cells to resist gemcitabine-induced lethality. They screened cellular structure with both DECIPHER Library Modules 1 and a couple of which target approximately 10,000 human genes in whole. Each lentiviral-based library module consists of 27,500 shRNA expression constructs targeting approximately 5,000 human genes—each gene targeted by 5-6 hairpins.

To run the screens, cells were transduced while using libraries then helped by gemcitabine with the goal to name genes that, when knocked down, made the cell weaker to gemcitabine. Interference using the function of those genes, then, would seem to become synthetically lethal with gemcitabine. Thus, these genes could be good potential targets for the combination treatment while using drug.

Inside the screening, they identified about 70 genes with synthetic lethal effects in conjunction with gemcitabine. Highly represented inside the hits were genes interested in DNA damage response and repair, which was expected since gemcitabine is often a DNA damaging agent. They focused specifically on genes identified within the screen that had been upstream from the checkpoint kinase 1 (CHK1) of the ATR/CHK1 pathway. Specifically, RAD17, HUS1, WEE1, and RFC3 all resulted in within the screen, and but RCF3 were subsequently confirmed with 3 independent shRNA constructs.

All of those other study dedicated to the RAD17 gene. They demonstrated that knocking down this gene increased lethality of gemcitabine by forcing cells with damaged DNA to get in mitosis. The same effect was shown previously for WEE1 by Aarts, et alii., that is also identified from the screen. In point of fact, a potent inhibitor of WEE1 kinase (MK-1775) is scheduled in conjunction with gemcitabine and platinum-based drugs for phase I clinical tests.

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Gene Cloning and Protein Expression

For your current PSI-2 target ORFs, boundaries defined by domain family analysis and some constructs are generated per target to optimize the means to get a successful outcome. Our permutation strategy will depend on extension with the N or C-termini depending on secondary structure, length restrictions or experimental data. When possible however, we attempted cloning and expression of the original full-length-target sequence. A lot of targets represent the MSCG assigned Pfam groups with approximately 25% of the target group composed of biomedical targets from pathogens. For that expressed target group, we observed a total rate of success of ~25% for generation associated with an expression clone that produced a soluble protein product sufficient for crystallographic studies.
cloning.02
In order to meet the fabrication goals for PSI-2, we apply a wide 96-well-plate HTP technology to come up with clones and express soluble proteins. Our pipelines use pMCSG7 because the primary expression vector along with a maltose-binding protein (MBP) fusion vector to get a “salvage” technique for proteins that express well in pMCSG7 but show low solubility. Expression clones that produce insoluble proteins are directed to Level 2 processing (see Figure). The developmental goal is to address solubility problems using HTP approaches. Criteria for entry to the salvage loop would include: deficit of a soluble orthologues, poor diffraction quality crystals, or high target priority due to biomedical impact. This tiered strategy leverages our efficient and cost-effective parallel processes suitable for mass production of proteins and protein fragments in E. coli.

Coding regions are amplified using primers made with the Express Primer tool or domain-specific primer design tools. All primers contain ligation-independent cloning sites compatible with multiple vectors. Affinity tags having a TEV (tobacco etch virus) protease cleavage site are fused to any or all proteins to facilitate their purification or capture. The main steps on the process — PCR gene amplification, testing for protein expression and solubility — are conducted in 96-well-plate format. Denaturing PAGE analysis of proteins is performed in a high-density gel format.
Cloning_strategy_28
In the event the PSI pilot centers were formed, ligation-independent cloning (LIC) offered a stylish technology adaptable for robotic cloning, but existing vectors weren’t suited to automated purification of proteins for crystallization. We developed a list of superior LIC vectors tailored tailored for this purpose. The vector, pMCSG7, encodes a His6-tag followed by a spacer and a TEV protease cleavage site that overlaps while using LIC site. This design puts the TEV site near to the start of the cloned native protein. Only the three-amino-acid-sequence SerAsnAla

If the PSI pilot centers were formed, ligation-independent cloning (LIC) offered a lovely technology adaptable for robotic cloning, but existing vectors just weren’t made for automated purification of proteins for crystallization. We developed a pair of superior LIC vectors tailored designed for this purpose. The vector, pMCSG7, encodes a His6-tag then a spacer and also a TEV protease cleavage site that overlaps using the LIC site. This design puts the TEV site close to the start of the cloned native protein. The three-amino-acid-sequence SerAsnAla (SNA) is added to the protein after protease cleavage.

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Another Group Finds Similar Keys to Optimal Pooled shRNA Library Screens

Our group recently ran across an article describing an independent RNAi screen with a non-Cellecta pooled shRNA expression library that piqued our interest. In the October 2011 online Genome Biology Journal, Sims, et al. comprehensively described how to run a rigorous genome-wide pooled RNA interference screen using next generation sequencing. The article thoroughly describes the procedural steps involved in screening a heterogeneous pooled library of thousands of lentiviral shRNA expression constructs. Although they used a library somewhat different than our design (the lack of unique sequenceable barcodes being one notable difference), the study nicely demonstrates many of the requirements to ensure meaningful screening results and emphasizes the need to use high throughput next-generation sequencing (as opposed to microarray hybridization) for reproducible measurements of shRNA depletion or enrichment following selection.

Viability or “drop-out” screens that look for depletion of shRNA sequences in selected populations to identify essential genes are one of the most common applications of pooled shRNA screening. The Sims et al. study focuses primarily on the key factors to ensure reproducible results for these screens. Among the most important ones, they note the following:

The shRNA expression library itself must be generated systematically to minimize variation in hairpin representation. This should be assessed by HT sequencing of the plasmid form of the library. Interestingly, Sims et al. also found that the plasmid library is a better reference for starting hairpin representation than the pseudoviral packaged library, which is consistent with our experience at Cellecta, too.

It is essential to manage cell numbers to maintain hairpin representation through the whole screen. Specifically, Sims et al. recommends maintaining at least 1,000 cells per RNA–which is also the ratio we find optimal as described in an earlier blog post. They also caution against letting cells grow past 70% confluency before replating.

Following selection, it is important to amplify sufficient genomic DNA to ensure a representative population from each cell sample. For their library of 10,000 shRNAs, they used at least 60ug of genomic DNA for pre-sequencing PCR amplification. We too find similar amounts necessary (i.e., for 27,000 shRNA, we us 200 ug/sample).

Biological replicates are a requirement to overcome stochastic noise inherent in the screen. However, replicates should have a high level of reproducibility with R-squared values of 0.9 or better.

The pooled shRNA library must be a reasonable size to enable practical handling of the cell populations, genomic DNA amplification, and biological replicates required for an effective screen. Sims et al used a library with 10,000 shRNA.

As a result of the thorough technique, Sims et al. estimated they were able to identify more than 98% of the hairpins in all replicates. One distinct difference in the Sims et al. library compared with Cellecta’s is the presence of an barcode, that is, a unique readily identifiable sequence separate from the hairpin sequence that can be used to identify the particular shRNA in the expression cassette. Somewhat confusingly, though, Sims et al. used the term “barcode screen” although no barcode is present in their library. Detection of shRNA levels in selected populations was done by sequencing a portion of the shRNA encoding region. From our experience, use of a separate unique barcode optimized for sequence analysis increases sequencing calls and helps improve replicate correlations. Sims et al. did find that the pre-sequencing PCR step introduced a certain amount of noise in the data, which is consistent with amplification variability of shRNA sequences as opposed to short standardized barcodes.

The consistency of the general findings of this independent study with our experience, however, is very encouraging. Using a similar but distinct library, Sims et al. have uncovered many of the same critical requirements for optimal screening with complex shRNA pools as we have. This alignment emphasizes the importance of these procedural details to obtaining meaningful screening results, and it provides additional support for RNAi screening standards of practice that was the topic of the previous post.

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Construction of a Stable Replicating Shuttle Vector for Caldicellulosiruptor species: Extending Genetic Methodologies to other Members of This Genus

University of Georgia researchers have developed a replicating shuttle vector based on a small plasmid for Caldicellulosiruptor bescii. The entire plasmid was cloned into an E. coli cloning vector. The shuttle vector enables modification of several members the genus (including C. hydrothermalis) providing them with features that are desirable to improve biomass-to-biofuel conversion. There was no evidence of DNA rearrangement during transformation and replication in C. bescii.

Biomass is a renewable resource that has shown promise to replace petroleum-based fuels while reducing greenhouse gas emissions. A key challenge towards achieving an economically viable biomass solution is that plants have built up a natural protection (or recalcitrance) against being converted to fuel. Therefore, more effort using special enzymes and microbes is needed to convert biomass into ethanol. In order to become more widely adopted, the high cost for biomass processing needs to be reduced.

A special type of bacterium, Caldicellulosiruptor bescii, has a high affinity for decomposing lignocellulosic biomass that includes agricultural residues such as rice straw, switchgrass, as well as hard- and softwoods. However, the products of C. bescii’s degradation of biomass are not effective as fuels. That is, these naturally occurring processes do not produce compounds of great economic interest. However, given its high affinity for decomposition of, and its ability to grow on, untreated biomass, C. bescii makes an attractive candidate for genetic modifications that could better enable its use in the production of biofuels and other commodity chemicals.

University of Georgia researchers have developed a genetic tool that allows for transformations of Caldi, making it a more viable organism for the degradation of recalcitrant biomass and production of biofuels and commodity chemicals. The researchers have leveraged the special properties of thermophiles, organisms that grow at relatively high temperatures, which lead to better biomass conversion. The Caldicellulosiruptor genus is the most thermophilic cellulolytic microbes known, and therefore this genus was targeted towards improving its efficiency for biomass conversion.

To improve the effectiveness of the Caldicellulosiruptor species, University of Georgia researchers constructed a replicating shuttle vector that enables advanced characterization and manipulation of genes and metabolic pathways to enhance biomass conversion. This is the first vector of its kind for members of this genus. Their technique also utilizes a native plasmid in this genus to generate a replicating vector.

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New knowledge about permafrost improving climate models

Climate

New research findings from the Centre for Permafrost (CENPERM) at the Department of Geosciences and Natural Resource Management, University of Copenhagen, document that permafrost during thawing may result in a substantial release of carbon dioxide into the atmosphere and that the future water content in the soil is crucial to predict the effect of permafrost thawing. The findings may lead to more accurate climate models in the future.

The permafrost is thawing and thus contributes to the release of carbon dioxide and other greenhouse gases into the atmosphere. But the rate at which carbon dioxide is released from permafrost is poorly documented and is one of the most important uncertainties of the current climate models.

The knowledge available so far has primarily been based on measurements of the release of carbon dioxide in short-term studies of up to 3-4 months. The new findings are based on measurements carried out over a 12-year period. Studies with different water content have also been conducted. Professor Bo Elberling, Director of CENPERM (Centre for Permafrost) at the University of Copenhagen, is the person behind the novel research findings which are now being published in the internationally renowned scientific journal Nature Climate Change.

“From a climate change perspective, it makes a huge difference whether it takes 10 or 100 years to release, e.g., half the permafrost carbon pool. We have demonstrated that the supply of oxygen in connection with drainage or drying is essential for a rapid release of carbon dioxide into the atmosphere,” says Bo Elberling.

Water content in the soil crucial to predict effect of permafrost thawing

The new findings also show that the future water content in the soil is a decisive factor for being able to correctly predict the effect of permafrost thawing. If the permafrost remains water-saturated after thawing, the carbon decomposition rate will be very low, and the release of carbon dioxide will take place over several hundred years, in addition to methane that is produced in waterlogged conditions. The findings can be used directly to improve existing climate models.

The new studies are mainly conducted at the Zackenberg research station in North-East Greenland, but permafrost samples from four other locations in Svalbard and in Canada have also been included and they show a surprising similarity in the loss of carbon over time.

“It is thought-provoking that microorganisms are behind the entire problem – microorganisms which break down the carbon pool and which are apparently already present in the permafrost. One of the critical decisive factors – the water content – is in the same way linked to the original high content of ice in most permafrost samples. Yes, the temperature is increasing, and the permafrost is thawing, but it is, still, the characteristics of the permafrost which determine the long-term release of carbon dioxide,” Bo Elberling concludes.

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