Single Nucleotide Polymorphisms (snps) on Poor Quality or Low Concentration dna samples Emily Antonides, November 29, 2012



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Single Nucleotide Polymorphisms (SNPs) on Poor Quality or Low Concentration DNA Samples
Emily Antonides, November 29, 2012
The most frequent occurring manifestation of genetic variation in the human genome is single nucleotide polymorphisms (SNPs). SNP, pronounced “snip”, is a small genetic variation that occurs within a person’s DNA sequence. The variation occurs with a single nucleotide, such as an A (adenine) replacing one of the other three nucleotides C (cytosine), T (thymine), or G (guanine), that make up the genetic code. About one out of every 1,000 nucleotides in the human genome is expected to be a SNP site, which makes up for more than 90% of all the differences between humans. These variations can either be harmless, just the difference in a person’s phenotype, or they can be harmful, they code for a disease such as diabetes or cancer. Single nucleotide polymorphisms are detected by using Real-Time PCR, microarrays, pyrosequencing, fluorescence homogenous assays, mass spectrometry, nucleotide extension, cleavage, or oligonculeotide ligation.
Real-Time PCR is the most used technology for detection of SNPs because it only requires a small amount of purified DNA, but however sometimes samples can contain low numbers of cells or poor quality DNA from suboptimal sources which make detection difficult. Catsburg et al. (2007) conducted an experiment using dried blood samples and DNA traces from plasma in which they added a pre-amplification step before doing the SNP analysis with Real-Time PCR. Six SNPs in the Mannose Binding Lectin 2 (MBL2) gene were chosen as targets for analysis. DNA was extracted from plasma from mothers and their newborn babies and from dried blood samples that were 3-5 years old. A forward primer FMBL2 and a reverse primer RMBL2 were used in the pre-amplification prior to the Real-time SNP analysis. It was possible to analyze all plasma samples and 93% of the old dried blood samples. Amplification prior to Real-Time PCR SNP analysis is a convenient, cost effective, and useful method to improve the reliable SNP detection in specimens that do contain very little or poor quality DNA from suboptimal sources.

References:
Catsburg, Arnold, Wil C. Van Der Zwet, Servaas A. Morré, Sander Ouburg, Christina M.J.E. Vandenbroucke-Grauls, and Paul H.M. Savelkoul. "Analysis of Multiple Single Nucleotide Polymorphisms (SNP) on DNA Traces from Plasma and Dried Blood Samples." Journal of Immunological Methods 321.1-2 (2007): 135-41. Print.
"MBL2." Genetics Home Reference. A Service of the U.S. National Library of Medicine, 19 Nov. 2012. Web. Nov. 2012. .
"SNPs: Variations on a Theme." NCBI: A Science Primer. N.p., 20 Sept. 2007. Web. Nov. 2012. .

Plantibodies: Tobacco-Derived Human Immunodeficiency Virus-Neutralizing Antibody
Mackenzie Owen, November 29, 2012

The Human Immunodeficiency Virus (HIV) affects millions of people (roughly 60 million in 2010). Extensive research has been conducted for more than twenty years on finding an effective vaccine against HIV. Even after all of that research, no effective vaccine has been produced. Because of the failure in finding a suitable vaccine, the focus of the research has been shifted to prevention strategies. Antibodies are predominantly produced using mammalian cells, such as the Chinese hamster ovary (CHO) cells, but transgenic plants have been used more and more. The elastin-like peptide (ELP) fusion technology has improved the stability of the plant-produced, recombinant proteins while assisting in their purification. This makes plants an alternative method, instead of mammalian cells, for producing antibodies.



An experiment by Floss et al. (2009) uses transgenic tobacco plants to produce the monoclonal antibody mAbs 2G12 and elastin-like peptide (ELP) fusion proteins. The transgenic tobacco plants accrued the four different formats of the anti-HIV-1 antibody, 2G12, in the endoplasmic reticulum. The kinetic binding parameters of the four formats of the 2G12 were identical to the 2G12 in CHO cells that were lacking the ELP fusion proteins when the affinity-purified antibodies were analyzed using surface plasmon resonance spectroscopy. Using inversion transition cycling to purify the proteins from the tobacco seeds did not affect the binding kinetics of the protein and antibody. Analysis of the N-glycans in the heavy chains from the leaf-derived antibodies showed that all four formats of the 2G12 were efficiently retrieved by the ER. The N-glycans in the seeds were seen localized to different subcellular departments. This experiment showed that the in vitro HIV-neutralization properties of the 2G12 derived in the tobacco was just as efficient as or more efficient than the CHO-derived 2G12.
Plantibodies and edible vaccines are becoming a more popular approach, instead of using more costly and less stable mammalian cells. Another advantage to producing plantibodies is that the technology is already available for processing plants. Also, using edible vaccines eliminates the purification requirement of the antibody produced in the plant.

References:

  1. Daniell, Henry, Stephen J. Streatfield, and Keith Wycoff, 2001. “Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants.” TRENDS in Plant Science Vol. 6 No. 5, May 2001.

  2. Floss, Doreen M., Markus Sack, Elsa Arcalis, Hohannes, Stadlmann, Heribert Quendler, Thomas Rademacher, Eva Stoger, Jügen Scheller, Rainer Fischer, and Udo Conrad, 2009. “Influence of elastin-like peptide fusions on the quantity and quality of a tobacco-derived human immunodeficiency virus-neutralizing antibody.” Plant Biotechnology Journal (2009) 7, pp. 899-913.


miRNA Profiling to Distinguish Bodily Fluids
Rachel Markert, November 29, 2012
This presentation is about identifying bodily fluids in forensic cases with the use of microRNA (miRNA). By using miRNA, it is hopeful that it will become a method commonly used in forensic labs because it is cheaper and easier than other methods used already. miRNAs are also useful because they are more stable due to being smaller than mRNA which is larger and is easier to degrade. This can be a problem for forensic scientists who work with degraded samples often.
In many forensic cases, bodily fluids are found at the scene of the crime and on victims. This leads to unknown fluids that need to be identified and connected to a suspect. This particular experiment only looked at blood and saliva. There are other bodily fluids collected as well, such as semen and vaginal secretions, common in rape cases. This is prompting more studies into these other bodily fluids. Blood and saliva have been found to contain mRNAs along with miRNAs. By using miRNAs, bodily fluids can be identified easier than other used methods and is also cheaper. This is what forensic labs are looking for in finding new methods to identify compounds. MicroRNA or miRNA is small noncoding RNA used in gene expression. They help to regulate expression and are useful in many cellular, posttranslational processes. The miRNA binds to mRNA and inhibits protein function.
Bodily fluids were collected to be used in microarrays, PCR verification, and to create an aged blood sample. RNA was extracted from the samples and then enriched for miRNAs by using kits provided by certain companies. A microarray was used next with reverse complement miRNA probes from human sequences already known. This created positive results for blood and saliva which were selected. These selected samples had Real-Time PCR performed to select for blood and saliva separately. A cDNA was created and used for quantitative PCR. A hierarchical clustering was created by performing mathematical equations.
By looking at the graphs and figures, it was shown that blood and saliva could be distinguished from the miRNA. Aged blood was also distinguished and proved that the miRNAs did not degrade during the aging process. The goal now is to find other processes that could distinguish bodily fluids that are highly degraded by natural elements such as wind, rain, and sunlight. It is also beneficial to look into other bodily fluids as well to be able to distinguish a higher number of them in forensic cases. In order for this method to be used in forensic labs, it must be standardized and proven to work.

References:

  1. Courts, C; Madea, B. “Specific micro-RNA signatures for the detection of saliva and blood in forensic body-fluid identification.” J Forensic Sci. v.56 no. 6 p. 1464-1470. 2011.

  2. Landgraf , P; Rusu , M; Sheridan , R; et al. “A mammalian microRNA expression atlas based on small RNA library sequencing.” Cell. v. 129 no. 7 p. 1401–14. 2007.

The Use of Chimeric Antigen Receptor-Modified T Cells to Aid in Leukemia Treatment

James Stockwell, November 29, 2012


Chronic Lymphoid Leukemia (CLL) is type of leukemia affecting B cell lymphocytes. In cases of CLL, the B cells grow out of control and eventually crowd out the normal B cells. Typically, CLL is first detected when white blood cell counts become abnormally high. Average survival rates depend on the type of mutation causing the abnormal replication, but range from 8 to 25 years. The case study described by Porter et al. (2011) demonstrates a treatment regime based around engineering T cells to eradicate B cells such that they may cure the cancer.

In order to program T Cells to eradicate CLL cancer cells a lentiviral-derived plasmid was engineered to express a chimeric protein derived from a number of proteins. First is the antigen specific portion of an antibody. In this instance anti-CD19 was chosen as it is specific to the CLL cancer cells and normal B cells. This is then combined with the intracellular domain CD3-zeta to be expressed as a single protein. Finally, in order to increase persistence of the T cells and increase their antitumor function, the signaling domain of CD137 was included in the chimeric protein. Prior to this case study, the effects of the inclusion of the CD137 signaling domain had only been demonstrated in vivo. This chimeric protein was placed into a lentiviral backbone in preparation for the transformation of patient T Cells.



The ClinicalTrials.gov number NCT01029366 clinical trial demonstrates the effect of patient T Cells modified with the vector previously described. The initial patient was diagnosed with CLL in 1996. In 2002, the patient began treatment with rituximab and fludarabine. Subsequently, treatments were received in 2006 and 2009. The treatment in 2009 began to show decreased effectiveness with greater number of cells showing mutations indicative of cancer after the treatment. In late 2009 T cells from the patient were collected, transformed with the anti-CD19 chimeric protein expressing lentiviral vector, propagated, and injected back into the patient over the course of three individual treatments.

Antitumor activity of the engineered T Cells was much greater than expected, and eventually led to tumor lysis syndrome in the patient. Hospitalization prevented long term damage from the acute kidney damage of the tumor lysis syndrome. Despite the adverse reaction, CLL related cells and normal B cells were undetectable 10 months after treatment.


References:

1. Porter D, Levine B, Kalos M, Bagg A, June C. Chimeric Antigen Receptor–Modified T Cells in Chronic Lymphoid Leukemia. N Engl J Med 2011; 365:725-733

2. Kalos M, Levine BL, Porter DL, et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med 2011;3:95ra73-95ra73


  1. Gross G, Waks T, Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Proc Natl Acad Sci U S A 1989;86:10024-10028

  2. Milone MC, Fish JD, Carpenito C, et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther 2009;17:1453-1464

  3. Irving BA, Weiss A. The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways. Cell 1991;64:891-901

Production of Engineered Long-Life, Male Sterile, and Pollen Free Pelargonium Plants

Stella Tamesis, November 29, 2012
Pelargonium is a genetically modified garden plant and is considered as one of the most popular and is of great importance in the plant market. Methods of gene transfer via Agrobacterium tumefaciens is used to improve the Pelargonium by introducing new traits/genes to the plant. These new traits that were introduced to the plant allowed the plant to breed a wide range of flowers, leaf shapes, scents, and even the ability to resist pest and diseases. The Agrobacterium tumefaciens, which causes crown gall disease, was used to carry these altered genes. One gene that encodes an enzyme called isopentenyl phosphotransferase or ipt is used to increase the amount of cytokinin and therefore prevents the aging process. Another is engineered to destroy any potential pollen-producing anthers. Therefore you get a genetically modified plant that can live a long time and is pollen free. For the genetic transformation of a long-life and male sterile Pelargonium zonale plants, the pSAG12::ipt and PsEND1 barnase chimaeric genes were used. Results show that using pSAG12::ipt delayed the senescence process and changed the architecture of the plant. PsEND1 barnase chimaeric genes created pollen free plants. Some methods used involve PCR analysis, Real time PCR, and light microscopy.

References:

BioMed Central Limited (2012, August 29). No more sneezing: Allergen-free house plants. ScienceDaily. Retrieved November 18, 2012, from http://www.sciencedaily.com /releases/2012/08/120831083315.htm


BioMed Central Limited (31 August 2012). Production of engineered long-life and male sterile Pelargonium plants. [Research Article] Available at: http://www.biomedcentral.com/1471-2229/12/156 . [Last Accessed 18 November 2012].
Biomedcentral.com (2012) www.biomedcentral.com - Figure. [online] Available at: http://www.biomedcentral.com/1471-2229/12/156/figure/F5 [Accessed: 27 Nov 2012].
Maria Lis-Balchin, (2007). Geranium and Pelargonium: The Genera Geranium and Pelargonium. 1st ed. New York: Taylor & Francis.

Mary Spink. Growing Pelargoniums. [ONLINE] Available at: : http://www.swanlandnurseries.co.uk/culturalnotes.asp. [Last Accessed 18 November 2012].


Scielo.cl (2007) Electronic Journal of Biotechnology - Influence of 2,4-D and BAP on callus growth and the subsequent regeneration of somatic embryos in long-term cultures of Pelargonium x domesticum cv. Madame Layal. [online] Available at: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582007000100007 [Accessed: 18 Nov 2012].

Green Fluorescent Protein

Samantha Stickdorn, December 4, 2012

Green fluorescent protein (GFP) is a protein found in the umbrella of Aequorea victoria jellyfish. It was discovered by Osamu Shimoura in 1962. This protein fluoresces bright green when exposed to ultra violet light and has a unique cylindrical beta barrel structure that protects its chromophore. Since its discovery, GFP has become a very important tool in biological research because it has so many fascinating applications and can be inserted into almost any organism from bacteria to insects and even mammals.

GFP can be used as a reporter gene or a tracer molecule. It can be used to tag proteins in order to track their movement and functions in living organism. Structures that would otherwise be invisible can be easily seen when labeled with GFP. This remarkable protein has been used to study everything from the function of neurons to the growth of cancer cells. GFP can be placed under the control of a promoter to determine the function of an unknown protein. Discoveries of mutations have made it possible to enhance the brightness of GFP and make fluorescent proteins in every color imaginable. These discoveries have only furthered the possibilities of these proteins.

References:



  • "Fluorescent protein technology." Zeiss microscopy online. Carl Zeiss Microscopy, 2012. Web. 30 Nov 2012.

  • marc, Zimmer. "Green Fluorescent Protein." . Connecticut college, 3 2012. Web. 30 Nov 2012. .

  • Shimomura, O., Johnson, F. H. and Saiga, Y. (1962), Extraction, Purification and Properties of Aequorin, a Bioluminescent Protein from the Luminous Hydromedusan, Aequorea. J. Cell. Comp. Physiol., 59: 223–239. doi: 10.1002/jcp.1030590302

  • Chalfie, M. (1995), GREEN FLUORESCENT PROTEIN. Photochemistry and Photobiology, 62: 651–656. doi: 10.1111/j.1751-1097.1995.tb08712.x

Nanoparticle mediated delivery of genetic material to plants

Isha Shrivastava, December 4, 2012
Nanotechnology is not a separate scientific field, but a new platform for the various existing disciplines including chemistry, physics, biology, biotechnology, information technology, neuroscience, and engineering. Nanoparticles that are being used in the plant biology can be manufactured by top-down or bottom-up approaches depending on the type of application being used for. For altering the gene expression, it is important to deliver genetic material such as DNA and small interfering RNA for producing virus, herbicide, stress, pathogen, and pest resistant strain of crops. The Traditional gene delivery systems face the problems of targeting delivery systems, transportation through the cell membrane, uptake and degradation in the cell endolysosomes, and intracellular transportation of DNA to the nucleus. Also, the viral gene delivery vectors have narrow host range and allow limited size of genetic material. The application of nanoparticles to deliver the genetic material not only increases the efficiency but also makes it applicable to both monocotyledonous and dicotyledonous plants with tissue specificity.

Vijayakumar et al. (2010) demonstrated that nanoparticles had lower plasmid and gold requirement as compared to the micrometer sized gold particles, and also reduced cellular damage providing higher chances of plant regeneration. Various groups have demonstrated the use of surface functionalized silica nanoparticles into animal cells and tissues. The main hindrance in applying this principle to plant cells was the presence of plant cell walls; however, Torney et al. (2007) initiated the use of a honeycomb mesoporous silica nanoparticle system that can transfer DNA and chemicals into isolated plant cells and intact leaves. In case of the mesoporous silica nanoparticle system (MSN), the drugs and imaging agents can be incorporated in the mesopores with covalently bound caps that prevents the drugs from leaching. The MSN can deliver DNA by using a plasmid containing a GFP under the control of a constitutive promoter.

Naqvi et al. (2011) have demonstrated the use of calcium phosphate mediated genetic transformation where the nanoparticles of size up to 100 nm are taken up by the cells by fluid-phase endocytosis and the released gene can cross the nuclear membrane by overcoming the nuclear pore complex barrier due to the presence of calcium ions. Hence, the delivery of genetic material in the plant cells by nanoparticles serves as an efficient, cost effective and non-destructive method that will definitely lead to advancements in plant cell and plant genomic manipulation applications and research.
References:

Ghormade, V., Deshpande, M.V., and Paknikar , K.M. 2011. Perspectives for nano-biotechnology enabled protection and nutrition of plants. Biotechnology Advances 29: 792-803

Husaini,A.M., Abdin, M.Z. , Parray, G.A. , Sanghera, G.S. , Murtaza, I. , Alam, T. , Srivastava, D.K. , Farooqi, H. , and Khan, H.N. 2010. Vehicles and ways for efficient nuclear transformation in plants. Landes Bioscience 1(5) : 276-287

Nair, R., Varghese, S.H. , Nair, B.G. , Maekawa , T. , Yoshida , Y. , and Kumar, D. S. 2010. Nanoparticulate material delivery to plants. Plant Science. 179: 154-163

Naqvi, S., Maitra , A.N. , Abdin, M. Z., Akmal, M. , Arora , I., and Samim, M. 2012. Calcium phosphate mediated genetic transformation in plants. Journals of Materials Chemistry 22: 3500 – 3507

Torney, F. , Trewyn, B. G. , Lin, V.S. , and Wang, K. 2007. Mesoporous silica nanoparticles deliver DNA and chemicals into plants. Nature Nanotechnology. 2: 295-300

Vijayakumar, P.S., Abhilash, O.U. , Khan, B.M., and Prasad B.L.V. 2010. Nanogold-loaded sharp edged carbon bullets as plant-gene carriers. Advanced Functional Materials. Plant Science. 20(15): 2416-2423

The Use of Transgenic Potatoes as a Vaccine against Cholera

Abigail Skorupski, December 4, 2012
Cholera is an infection in the small intestine caused by the bacterium Vibrio cholerae. Symptoms of cholera include watery diarrhea and vomiting, leading to rapid dehydration and even death. The cholera toxin is made up of one A subunit and five B subunits. The A subunit is the enzymatically active portion, and is responsible for the loss of water. The B subunit is enzymatically inactive, and binds GM1-gangliosides on the surface of intestinal epithelial cells. Because the B subunit is inactive, it is a good candidate for a subunit vaccine because it can elicit an immune response without being toxic to the host. Because oral vaccination is more effective than parental vaccination against pathogens that invade through mucosal surfaces, the consumption of transgenic potatoes containing the CTB gene is a practical way to protect humans against cholera. Furthermore, oral vaccines are more practical than traditional vaccines because they are cheaper, safer, and easier to administer.

Arakawa, et al. (1997) conducted an experiment to transformed potato leaf explants with a vector containing the CTB gene encoding the cholera toxin B subunit protein, fused to an endoplasmic reticulum retention signal. The explants were transformed by Agrobacterium tumefaciens and the fusion gene was identified in the genomic DNA of bioluminescent plants by PCR amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein. The maximum amount of CTB protein detected in potato leaf and tuber tissues was approximately 0.3% of the total soluble plant protein. Furthermore, they determined that in the presence of the ER retention signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties.

Arakawa, et al. (1998) tested the production of antibodies in mice against transgenic potato containing the CTB gene. Both serum and intestinal CTB-specific antibodies were induced in orally immunized mice. Furthermore, the cytopathic effect of cholera holotoxin (CT) on Vero cells was neutralized by serum from mice immunized with transgenic potato tissues. Following intraileal injection with CT, the plant-immunized mice showed up to a 60% reduction in diarrheal fluid accumulation in the small intestine.

Finally, Tacket, et al. (1998) conducted a human clinical trial to determine the amount of antibodies generated in humans against the transgenic potato containing the CTB gene. Fourteen healthy adult volunteers ingested 100g of transgenic potato, 50g of transgenic potato, or 50g of wild-type potato. Researchers found that 91% of 11 volunteers who ingested transgenic potatoes and none of those who ingested wild-type potatoes developed 4-fold rises in IgG anti-LT. 73% of 11 volunteers who ingested the transgenic potatoes developed neutralization titers > 1:100. Finally, 50% of ten volunteers who ingested transgenic potatoes developed 4-fold rises in sIgA. These results offer a new strategy for developing safe and inexpensive vaccines against cholera.


References:

Arakawa T, Chong D, Langridge W. (1998) Efficacy of a food plant-based oral cholera toxin B subunit vaccine. Nature Biotechnology 16(3): 292-297.

Arakawa T, Chong D, Merritt J, Langridge W. (1997) Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Research 6(6): 403-413.

Tacket C, Mason H. A (1999) review of oral vaccination with transgenic vegetables. Microbes And Infection / Institut Pasteur 1(10): 777-783.

Tacket C, Mason H, Losonsky G, Clements J, Levine M, Arntzen C. (1998) Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nature Medicine 4(5): 607-609.

Hypoxia-targeted Gene Therapy of Tumors using Virus-directed Enzyme-Prodrug Systems

Jeff Voegele—December 4, 2012
Solid tumors are a result of more than 90% of all human cancers. These masses of rapidly dividing cells form from a single mutated cell, upon which the disease spreads to surrounding tissue. The growing tumor must obtain its own blood supply, and it achieves this by stimulating the growth of nearby blood vessels. This process is called angiogenesis. Tumor blood vessels are typically very irregular, which decreases the efficiency of oxygen delivery to the cancer cells. These cancer cells that are depleted of oxygen are known as hypoxic areas of the tumor. Hypoxic regions are more resistant to radiotherapy and chemotherapy than well-oxygenated (normoxic) areas. They are also related to malignant progression, increased invasion, angiogenesis, and increased risk of metastasis.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that responds to changes in oxygen levels within the cell. Overexpression of the α-subunit of this protein is thought to lead to increased tumor aggression. In hypoxic cells, HIF-1 activates transcription by binding to hypoxic-response elements (HREs) within the promoter regions of genes, leading to overexpression of specific proteins in the tumor. Two such proteins that are often overexpressed in tumors are vascular endothelial growth factor (VEGF) and erythropoietin (EPO). The HREs of these two proteins have been shown to be sensitive to hypoxia and can therefore be used in gene therapy to selectively target hypoxic cells for therapeutic gene expression. The aim of this research is to combine this hypoxia-targeted gene therapy with traditional cancer therapy methods for enhanced cell kill (cytotoxicity) of the tumor.

The use of a viral vector is a commonly used method to deliver a target gene for therapeutic purposes. In virus-directed enzyme prodrug therapy (VDEPT), a gene encoding a prodrug-activating (“suicide”) enzyme is delivered to the target tissue using a viral vector. A non-toxic prodrug is then administered, and the suicide enzyme metabolizes it into a toxic compound. The toxins are then free to diffuse to neighboring cells and kill them via the “bystander effect.” Two examples of these prodrug-activating genes are the Herpes Simplex Virus thymidine kinase (HSVtk) and bacterial nitroreductase (NTR), whose corresponding prodrugs are Ganciclovir (GCV) and CB1954, respectively.

Harvey et al. (2011) constructed plasmid vectors encoding HSVtk and NTR suicide genes, under control of either VEGF or EPO HREs combined with either the minimal cytomegalovirus (mCMV) or minimal interleukin-2 (mIL-2) promoter. The optimal hypoxia-inducible HRE-promoter combination was determined to be VEGFmCMV. The researchers then created adenoviral vectors of this optimal hypoxia-inducible promoter with the prodrug-activating enzyme genes (HSVtk and NTR), and compared the cytotoxic effects in established cancer cell lines and in a primary human ovarian cancer cell culture in vitro. After administration of the prodrugs, both transgenic systems showed significant cytotoxic effects in the cancer cell lines as well as in the cancer patient cultures. However, the NTR/CB1954 system was shown to be more effective, so it was used in further trials. In vivo testing was done in nude mice that contained transplanted HCT116 tumors (xenografts). An adenoviral vector containing the NTR transgene under control of the optimal hypoxia-inducible promoter (VEGFmCMV) was intratumorally injected into the HCT116 xenografts of the mice. Tumor volumes were then recorded daily over a period of 15 days as the CB1954 prodrug was administered. The Ad-VEGFmCMV-NTR vector showed a significant delay in tumor growth in the presence of CB1954, but not in its absence. Immunostaining experiments also verified the hypothesis that transgene expression under control of the hypoxia-inducible promoter was localized to hypoxic areas of the tumor.


References:

TJ Harvey, IM Hennig, SD Shnyder, PA Cooper, N Ingram, GD Hall, PJ Selby, and JD Chester. Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo. Cancer Gene Therapy (2011). 18, 773-784.

Brown, JM. Exploiting the hypoxic cancer cell: mechanisms and therapeutic strategies. Mol Med Today (2000) 6, 157-162.

Creation of a subunit vaccine against avian (H5N1) influenza

Steven Mitchell, December 4, 2012
Influenza is a disease that has plagued mankind for quite a long time. It is crafty in that it is constantly and rapidly mutating, making it difficult to control or eradicate. Currently, traditional vaccines are ineffective because of the rapid mutations and that culturing the virus is time-consuming and inefficient. Vaccines also have a limited shelf life, making them essentially unavailable to third-world countries that have no access to refrigeration. Recombinant DNA technology may hold the key to controlling this pathogen all across the globe. Targeting areas of low mutation, rDNA technology could theoretically create a universal vaccine designed to target all strands of influenza. Also, this technology could outperform traditional methods because of it’s low cost, expanded shelf life, and high yield and production rate.

Recombinant DNA technology could theoretically be much more effective at preventative medicine rather than traditional methods. This is because not all infectious agents can be grown in culture, so no vaccines have been developed for a number of diseases. Also, production of animal and human viruses requires animal cell culture, which can be expensive. Both yield and rate of production of animal and human viruses are usually low, which means vaccine production becomes costly. Safety is a very prominent issue with traditional methods; an example of safety issues is the level of containment that is needed when working with live viruses. Also, batches need to be checked multiple times to ensure that killed or attenuated strains will not inadvertently infect the vaccine recipients. Another issue with traditional methods is that certain diseases such as HIV cannot be prevented.

A study conducted by Du et al. (2011) examined the potential for creating a subunit vaccine against a highly pathogenic and deadly strain of avian influenza known as HPAI-H5N1. This virus is particularly difficult to produce a traditional vaccine because of the rapid mutation of hemagglutinin (HA) protein. This protein is present on the surface of all influenza viruses, but mutates readily so that antibodies have a difficult time recognizing new strains of H5N1. This limits traditional vaccines to targeting single strains, and limits the potential to cross-protective immunity. The gene for hemagglutinin was used to create a subunit vaccine against specific strains of H5N1, as well as divergent strains that could arise from mutation. The HA1 gene was used in a construct to prime the recipient’s immune system to recognize H5N1 hemagglutinin. The HA1 gene was chosen for expression rather than HA2 because HA1 remains hardly mutated throughout various strains of avian influenza. A protease cleavage site between HA1 and HA2 was fused to the HA1 gene with the gene that encoded for the Fc portion of an IgG antibody, as well as a gene that encoded a foldon protein. Both of these genes attached to the HA1 gene were implemented to increase proper folding conformation of the HA1 protein. This proper folding was necessary so that the correct IgG antibodies would be produced to stave off invading viruses.
References

Davidson, Michael. "The Influenza (Flu) Virus." Molecular Expressions. Florida State University, 28 2005. Web. 3 Dec 2012. .

Du, Lanying, Lanying Du, et al. "A Recombinant Vaccine of H5N1 HA1 Fused with Foldon and Human IgG Fc Induced Complete Cross-Clade Protection against Divergent H5N1 Viruses ." PLoS ONE. 6.1 (2011): 1-10. Print.

Glick, Bernard. Molecular biotechnology: principles and applications of recombinant DNA. 4th ed. Washington, DC: ASM Press, 2010. 459-497. Print.

Neumann, Gabriele, Hualan Chen, et al. "H5N1 influenza viruses: outbreaks and biological properties." Cell Research. 20.1 (2010): 51-61. Print.

Racaniello, Vincent. "Influenza Hemagglutination Assay." Virology Blog. N.p., 27 2009. Web. 3 Dec 2012. .



Generation of Bacterial Cells through Chemical Synthesis of the Genome

Curran Rhodes, December 6, 2012
As recombinant DNA technology has advanced it has become possible to manipulate the properties of bacterial cells through the insertion of individual genes into the pre-existing genome. However, there are several limitations on what functions can be achieved through these methods. DNA synthesis technology has also come a long way and it has become possible to design and synthesize DNA strands thousands of base pairs long. Researchers are beginning to the see that this technology can be used to create entire bacterial genomes.

Gibson et al. (2010) has demonstrated that bacterial cells controlled completely by a synthetically constructed genome can be constructed. They designed and synthesized a complete 1.08Mbp genetic sequence in yeast through homologous recombination of cassettes prepared by combining synthesized oligonucleotides. Three sets of recombination were used to sequentially combine the cassettes until the entire genome was produced.

The genome was modeled after the wild type sequence of M. mycoides with the exception that 4 watermark sequences were intentionally inserted to distinguish the artificial genome from the natural one. After extracting the synthesized DNA from yeast they transplanted the synthetic genome into the known bacterial strain M. capricolum to create a new strain that follows the genetic instructions designed and inserted by the investigators. Two dimensional gel electrophoresis revealed almost identical patterns of protein spots between the synthetic M. capricolum cells and wild type M. mycoides confirming that the artificial genome is being expressed. The protein expression of the synthetic cells did not resemble that of wild type M. capricolum.

In the future researchers hope that studies such as this one can serve as models for the design of entirely synthetic organisms. Not only have they shown that it is possible to transplant synthetic DNA into an organism to produce a new organism capable of self replication that follows the genetic instructions inserted by the researchers, but they have demonstrated that a competent artificial genome can be produced via sequence technology and homologous recombination. This has huge implications for future work into the synthesis of entirely novel microbes for biofuel production, waste management, and various other processes.


References:

Gibson, D; Glass, J; Lartigue, C; et al. “Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome.” Science. V. 329 p. 52-56. 2010.

Thomason, L; Court, D; Bubuneko, M; et al. “Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination.” Current Protocols in Molecular Biology. 78:1.16.1–1.16.24. (what year?)

“DNA Oligo FAQ.” Invitrogen Life Technologies. Available at: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Product-Types/Primers-Oligos-Nucleotides. Accessibility verified: December 2, 2012.



Dental Vaccines: The Production of Vaccines for the Treatment of Dental Caries

Scott Karas, December 6, 2012

ABSTRACT


A bacterium called Streptococcus mutans has been identified for the growth of mutans streptococci (MS) which causes dental caries or the more common name of cavities (1). MS has been known for being the main cause of dental caries in humans. Protein antigens from S. mutans have been identified to help with inhibiting the activation of sucrose-independent and sucrose-dependent mechanisms from binding to the tooth surface (1). Secretory IgA antibodies have been proven to prevent the activation of theses mechanisms. These type of antibodies are found and produced in saliva and secreted by mucous membranes (1). One example of this type of antibody is called salivary IgA antibody (1,2). These antibodies can be made into a subunit vaccine and be injected by the method of mucosal vaccination (1). A process that injects the vaccine into the mucous membrane and the goal is to use this type of vaccine on children or infants (1,2).

The sucrose-dependent cycle works by the binding of glucan to the tooth with the help of glucan binding protein B (GbpB) (2). Once glucan is bound MS secretes an enzyme, glucosyltransferase (GTF), to synthesize sucrose from glucan. The production of sucrose contributes to the growth of dental caries. Regulating the production of GbpB or GTF can result in the decrease in dental caries in humans (2). The production of synthetic peptides to mimic GbpB and GTF can help form a vaccine to create immunity from the MS pathogens (2).

REFERENCES

1. Russell, Michael W., Noel K. Childers, Suzanne M. Michalek, Daniel J. Smith, and Martin A. Taubman. "A Caries Vaccine?" ProQuest Nursing & Allied Health Source 38.3 (2004): 230-35. Print.

2. Smith, Daniel J., William F. King, Joy Rivero, and Martin A. Taubman. "Immunological and Protective Effects of Diepitopic Subunit Dental Caries Vaccines." Infection and Immunity 73.5 (2005): 2797-804. Print.

Not Your Ordinary Grass: Switchgrass as a Candidate for Cellulosic Ethanol

Avery Tucker, December 6, 2012

In 2007 the Renewable Fuels Standard was passed in order to increase the production the renewable biofuels ethanol. The act mandates that diesel and gasoline be blended with an amount of ethanol based on yearly production. Initial mandates were larger than yearly production could keep up with and the entire program has been largely scaled back. Today, ethanol production for fuel use is largely derived from starch, especially that of corn. However, as corn is also a food commodity, ethanol production competes with food production thus making it a less desirable precursor crop. Cellulosic ethanol production is an alternative to methods that rely on the plant structural molecule cellulose, which is not readily digested by humans. It is conceivable to first harvest the grain or fruit from a crop followed by a secondary harvesting of any remaining biomass for cellulosic ethanol conversion or to cultivate non-food crops, such as switchgrass, in areas that are outside delimited food-crop boundaries


Several obstacles currently make cellulosic ethanol production expensive. The main obstacle comes from the separation of lignin and cellulose, which are essential components of the cell wall. This step requires heavy acids that break apart the lignin, a large and very complex molecule, from the cellulose. Transgenic engineering of plants is being undertaken to modify lignin content in plants. By modifying constituent genes it is possible to decrease lignin content and increase cellulose thereby reducing pretreatment costs. For example, in the paper “Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass” (Fu et al. 2011) one common approach for the reduction of lignin was assessed. Through the down-regulation of the switchgrass O-methyltransferease gene it is possible to generate plants with low lignin profiles. Although not a large reduction, testing of cellulosic ethanol yields showed a 38% increase in biomass, 300-400% lower treatment requirement of cellulase, and higher yields of ethanol after fermentation with Clostridium thermocellum without the addition of enzymes.

References

Bulls, Kevin. "What's Holding Biofuels Back?" MIT Technology Review. N.p., 06 Aug. 2010. Web. 04 Dec. 2012. .

Fu, C., J. R. Mielenz, X. Xiao, Y. Ge, C. Y. Hamilton, M. Rodriguez, F. Chen, M. Foston, A. Ragauskas, J. Bouton, R. A. Dixon, and Z.-Y. Wang. "Genetic Manipulation of Lignin Reduces Recalcitrance and Improves Ethanol Production from Switchgrass." Proceedings of the National Academy of Sciences 108.9 (2011): 3803-808.

Glick, Bernard R., Jack J. Pasternak, and Cheryl L. Patten. Molecular Biotechnology. Washington: ASM, 2010. Print.

Schmer MR, Vogel KP, Mitchell RB, Perrin RK. Net energy of cellulosic ethanol from switchgrass. Proceedings of the National Academy of Sciences of the USA. 2008;105:464–469.

Stricklen, Miriam B. "Plant Genetic Engineering for Biofuel Production: Towards Affordable Cellulosic Ethanol." Nature.com. Nature Publishing Group, June 2008. Web. 05 Dec. 2012.




Genetic Engineering of “Golden Rice”

Meng Li, Decenber 6, 2012

The intake of vitamin A provides humans with an important nutrient for vision, growth, reproduction, cellular differentiation and proliferation, and integrity of the immune system. Vitamin A deficiency can result in visual or ocular malfunctions such as night blindness and xerophthalmia and can reduce immune responsiveness, which can result in an increased incidence or severity of respiratory infections, gastrointestinal infections, and measles. Vitamin A can be obtained from food, either as preformed vitamin A in animal products or as provitamin A carotenoids, mainly beta-carotene in plant products. Rice is the major staple food for hundreds of millions of people. It is generally consumed in its milled form with outer layers (pericarp, tegmen and aleurone layers) removed. The main reason for milling is to remove the oil-rich aleurone layer, which turns rancid upon storage, especially in tropical and subtropical areas. As a result, the edible part of rice grains consists of the endosperm, filled with starch granules and protein bodies, but it lacks several essential nutrients for the maintenance of health, such as carotenoids exhibiting provitamin A-activity. To obtain a functioning provitamin A (-carotene) biosynthetic pathway in rice endosperm, they introduced in a single, combined transformation effort the cDNA coding for phytoene synthase (psy) and lycopene-cyclase (-lcy) both from Narcissus pseudonarcissus and both under the control of the endosperm-specific glutelin promoter together with a bacterial phytoene desaturase (crtI, from Erwinia uredovora under constitutive 35S promoter control). This combination covers the requirements for -carotene synthesis and, as hoped, yellow-carotene-bearing rice endosperm was obtained in the T0-generation. In a proof-of-concept study, this study has shown that it is possible to establish a biosynthetic pathway de novo in rice endosperm, enabling the accumulation of provitamin. In part, this involves the use of different structural genes and the use of different selectable marker genes. Studies on the bioavailability of the provitamin A, transfer of the trait into agronomically important varieties, and risk assessments will be carried out in collaboration with other research institutes.



References:

1. Peter Beyer, Salim Al-Babili, Xudong Ye and Ingo Potrykus, et al. 2002, Symposium: Plant Breeding: A New Tool for FightingMicronutrient Malnutrition, The Journal of Nutrition, 132: 506s-510S.

2. Guangwen Tang, Yuming Hu, Michael A Grusak, and Robert M Russell, et al., 2012, b-Carotene in Golden Rice is as good as b-carotene in oil at providing vitamin A to children, The American Journal of Clinical Nurtion, 96: 658-64.

3. Guangwen Tang, Gregory G Dolnikowski, , and Michael A Grusak, et al., 2009, Golden Rice is an effective source of vitamin A, The American Journal of Clinical Nurtion, 2009:89:1776-83



Phytoremediation and Phytosensing of Chemical Contaminants, RDX and TNT: Identification of the Required Target Gene
Ariel Moore, December 6, 2012
The goal of this experiment is to use phytosensing and phytoremediation of explosives like RDX and TNT. Phytosensing is the detection of contaminants using plants. Phytoremediation is a process of decontaminating the soil by using plants to absorb and break down the contaminants.  RDX is 1, 3, 5 – trinitro – 1, 3, 5 – triazine and TNT is 2, 4, 6 – trinitrotoluene. These two explosives typically remain in the biosphere for a long period of time and are a source of toxic, mutagenic, and carcinogenic effects on humans.

Arabidopsis thaliana were was grown in Murashige and Skoog medium supplemented with vitamin B5, one percent sucrose and two percent gelrite at a pH of 5.8. The RNA was repared on the Murashige and Skoog medium containing 0.5 mili-molar mM RDX or 2 micro molar μM TNT or no explosive but with proportional amounts of the solvent DMSO. Microarray analysis of these three media was conducted and the results were consistent with theories for xenobiotic metabolism of plants. Xenobiotic is a chemical or substance that is foreign to an organism or biological system. There were many genes upregulated and these upregulated genes could be used for phytosensing and phytoremediation of the contaminants from the explosives.
References

Panz, Katarzyna, and Korneliusz Miksch. "Phytoremediation of Explosives (TNT, RDX, HMX) by Wild-type and Transgenic Plants." Science Direct. N.p., n.d. Web. 28 Nov. 2012. .



Rao, Murali, and Et. Al. "Phytoremediation and Phytosensing of Chemical Contaminants, RDX and TNT: Identification of the Required Target Genes." Springer Linker. Springer-Verlag, 19 June 2009. Web. 29 Nov. 2012. .

Rylott, Elizabeth L., and Neil C. Bruce. "Trends in Biotechnology - Plants Disarm Soil: Engineering Plants for the Phytoremediation of Explosives." Science Direct. N.p., Feb. 2009. Web. 28 Nov. 2012. .


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