This document summarizes research aimed at developing a more efficient system for screening potential drug inhibitors of P-glycoprotein (P-gp), an ATP-binding cassette transporter that pumps chemotherapeutics out of multidrug-resistant cancer cells. The researchers introduced the human MDR1 gene, which encodes P-gp, into a bacterial plasmid and transformed E. coli cells to express the protein. Sequence analysis confirmed the MDR1 gene was present in the plasmid as designed. Developing this bacterial screening system could allow high-throughput screening of potential P-gp inhibitors to identify drugs that could combat multidrug resistance in cancer.
Data driven strategies and considerations for scalable purification of Plasmi...Merck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/2JeT1U9
Plasmid DNA (pDNA) presents unique manufacturing challenges. While research scale purification kits simplify small pDNA preparations, scalable manufacturing must leverage significant process understanding. This webinar presents scalable solutions for all downstream unit operations from harvest to bulk filtration.
Plasmid DNA (pDNA) has been an important scientific tool for decades, but as clinical and commercial applications increase, manufacturers of pDNA face pressure to optimize production activities to meet demand while maintaining critical quality attributes. Key challenges in pDNA manufacturing exist around purification unit operations due to its large size, high viscosity, shear sensitivity, and similarities between pDNA and impurities. Overcoming downstream challenges with scalable techniques requires in depth knowledge of unit operation parameters and holistic process understanding. Our work investigates parameters and key considerations for purification unit operations including harvest, lysis, clarification, tangential flow filtration, chromatography, and sterilizing grade filtration.
In this webinar, you will learn:
• Parameters for E. coli harvest using microfiltration tangential flow filtration
• Key considerations for scalable alkaline lysis
• Filter selection guidance for clarification of alkaline lysate
• Purification strategies using AEX chromatography resins and membranes
• Implementation considerations for ultrafiltration/diafiltration
• Watch-outs for sterile filtration
• Purification process flow for Plasmid DNA
Data driven strategies and considerations for scalable purification of Plasmi...Merck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/2JeT1U9
Plasmid DNA (pDNA) presents unique manufacturing challenges. While research scale purification kits simplify small pDNA preparations, scalable manufacturing must leverage significant process understanding. This webinar presents scalable solutions for all downstream unit operations from harvest to bulk filtration.
Plasmid DNA (pDNA) has been an important scientific tool for decades, but as clinical and commercial applications increase, manufacturers of pDNA face pressure to optimize production activities to meet demand while maintaining critical quality attributes. Key challenges in pDNA manufacturing exist around purification unit operations due to its large size, high viscosity, shear sensitivity, and similarities between pDNA and impurities. Overcoming downstream challenges with scalable techniques requires in depth knowledge of unit operation parameters and holistic process understanding. Our work investigates parameters and key considerations for purification unit operations including harvest, lysis, clarification, tangential flow filtration, chromatography, and sterilizing grade filtration.
In this webinar, you will learn:
• Parameters for E. coli harvest using microfiltration tangential flow filtration
• Key considerations for scalable alkaline lysis
• Filter selection guidance for clarification of alkaline lysate
• Purification strategies using AEX chromatography resins and membranes
• Implementation considerations for ultrafiltration/diafiltration
• Watch-outs for sterile filtration
• Purification process flow for Plasmid DNA
This is the Powerpoint presentation from my recent presentation at the TTP LabTech US Acumen Users Group Meeting (UGM) held at the British Consulate-General in Cambridge, MA on May 18, 2010
Unlocking the Potential of mRNA Vaccines and TherapeuticsMerck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3lNmkf7
The therapeutic potential of mRNA has been studied for decades and this exciting modality could potentially disrupt the biological market, in particular vaccine and novel therapies. This webinar will highlight the potential of mRNA therapies and focus on the manufacturing process's associated challenges, solutions and perspectives from synthesis to delivery.
mRNA has emerged as a promising modality for a wide range of therapeutics and vaccines and could become the break-through technology of this century. mRNA-based platform technologies could enable a more rapid response to infectious diseases, outbreaks or pandemics and allow efficient gene replacements or cancer treatments. mRNA represents a safer alternative to DNA-based therapies and the technology has recently advanced to overcome stability and efficacy challenges. Because of that, the industrialization of this technology is just in its infancy stages and bottlenecks exist around scalability, purity, and delivery which are key to establish and deliver the promise of such platform. This webinar will shed light on the potential of mRNA therapies and focus on the manufacturing process's associated challenges, solutions and perspectives from synthesis to delivery.
In this webinar, you will learn:
• The potential behind using mRNA as a therapeutic and vaccine
• The mRNA production process
• The challenges around mRNA production
• The solutions and perspectives for a robust manufacturing process
• mRNA delivery systems and their manufacturing
Epigenetic silencing of MGMT (O6-methylguanine DNA methyltransferase) gene in...arman170701
O6–methylgunine-DNA methyltransferace (MGMT) is a DNA binding protein that is involved in repairing mutations.
MGMT gene - a tumor suppressor gene that codes MGMT (O6-methylguanine DNA methyltransferase) protein.
The MGMT protein removes mutagenic methyl groups from guanines through the methyltransferase activity.
Peptide nucleic acid (PNA) is a synthetic analogue of nucleic acids (DNA & RNA) with unique characteristics and several potential applications in biotechnology and biomedicine. This presentation is a slide format of an 2020 review article from Yale university scientists focused on genome editing application of PNA in votro, ex vivo and in vivo.
This is the Powerpoint presentation from my recent presentation at the TTP LabTech US Acumen Users Group Meeting (UGM) held at the British Consulate-General in Cambridge, MA on May 18, 2010
Unlocking the Potential of mRNA Vaccines and TherapeuticsMerck Life Sciences
Watch the presentation of this webinar here: https://bit.ly/3lNmkf7
The therapeutic potential of mRNA has been studied for decades and this exciting modality could potentially disrupt the biological market, in particular vaccine and novel therapies. This webinar will highlight the potential of mRNA therapies and focus on the manufacturing process's associated challenges, solutions and perspectives from synthesis to delivery.
mRNA has emerged as a promising modality for a wide range of therapeutics and vaccines and could become the break-through technology of this century. mRNA-based platform technologies could enable a more rapid response to infectious diseases, outbreaks or pandemics and allow efficient gene replacements or cancer treatments. mRNA represents a safer alternative to DNA-based therapies and the technology has recently advanced to overcome stability and efficacy challenges. Because of that, the industrialization of this technology is just in its infancy stages and bottlenecks exist around scalability, purity, and delivery which are key to establish and deliver the promise of such platform. This webinar will shed light on the potential of mRNA therapies and focus on the manufacturing process's associated challenges, solutions and perspectives from synthesis to delivery.
In this webinar, you will learn:
• The potential behind using mRNA as a therapeutic and vaccine
• The mRNA production process
• The challenges around mRNA production
• The solutions and perspectives for a robust manufacturing process
• mRNA delivery systems and their manufacturing
Epigenetic silencing of MGMT (O6-methylguanine DNA methyltransferase) gene in...arman170701
O6–methylgunine-DNA methyltransferace (MGMT) is a DNA binding protein that is involved in repairing mutations.
MGMT gene - a tumor suppressor gene that codes MGMT (O6-methylguanine DNA methyltransferase) protein.
The MGMT protein removes mutagenic methyl groups from guanines through the methyltransferase activity.
Peptide nucleic acid (PNA) is a synthetic analogue of nucleic acids (DNA & RNA) with unique characteristics and several potential applications in biotechnology and biomedicine. This presentation is a slide format of an 2020 review article from Yale university scientists focused on genome editing application of PNA in votro, ex vivo and in vivo.
The Matrix metalloproteinase-9 is involved in several pathologies. Its strong presence in ocular pathologies explains our interest for its genetic variation in cataract, glaucoma and retinoblastoma in Senegal. MMP9 is highly polymorphic with cataract and glaucoma. 77 mutations were noted with 21 haplotypes for the entire population. The haplotype diversity Hd is 0.831 and the nucleotide diversity Pi is 0.016; k = 17.395. The polymorphism of the Matrix metalloproteinase-9 gene is associated with all three diseases and SNP 3918249 is found in both cataract and glaucoma.
Streamlined next generation sequencing assay development using a highly multi...Thermo Fisher Scientific
Next generation sequencing (NGS) assay development for solid tumor sequencing requires characterization of variant calling directly from formalin-fixed paraffin embedded (FFPE) tissue samples. However, cell line based FFPE and human FFPE samples only contain 2 to 20 variants, which require laboratories to invest significant resources in sample sourcing and preparation when developing assays to detect 100+ variants
Genotyping of 27 Human Papillomavirus Types by Using L1 Consensus PCR Product...Alberto Cuadrado
Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or
GP51/61) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype
determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for
substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled
PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis,
genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle.
Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized
to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albuminconjugated
oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on
this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55,
56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancerassociated,
genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of b-globin
probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the
performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al.,
p. 132–152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach,
(1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics,
Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92%
concordance for HPV positivity (kappa 5 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples
resulted from weak signals and can be attributed to sampling error from specimens with low concentrations
(<1 copy/ml) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly
genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of
misclassification.
Final CSPG4 Presentation Abhinav Bhaskar 4-22-15.pptx
SMU Poster
1. Niharika Choudhury1, Collette Marchesseault2, Dr. John Wise2, and Dr. Pia Vogel2
1The Hockaday School, Dallas, TX; 2 Department of Biological Sciences, Southern Methodist University, Dallas, TX
High-Throughput Screening System for P-glycoprotein Inhibition
Introduction
While chemotherapeutics have seen some success in eliminating cancerous
cells, they have seen much failure as well. The overexpression of P-
glycoprotein (P-gp) has led to multidrug resistant (MDR) cancer cells that
keep chemotherapeutics from producing effective cytotoxicity in tumor
cells.
The ATP-binding cassette (ABC) transporter P-glycoprotein serves as a
pump that transports cytotoxins across its plasma membrane. In doing so,
P-gp protects the cells in the body from foreign substances and contributes
to the effectiveness of the blood-brain barrier or liver in pumping out toxins
and drugs; however, cancer cells that express P-gp in large amounts are
rendered multidrug resistant, as they do not respond effectively to
chemotherapeutics. Anti-cancer drugs that enter the cells are merely
pumped out by the protein, and higher doses of the chemotherapeutic are
required until the patient can no longer withstand treatment due to the
toxicity.
In response, there has been ongoing research to find drug inhibitors that
keep P-gp from pumping out chemotherapeutics while maintaining
reasonable toxicity levels in the body. Dr. John Wise and Dr. Pia Vogel’s lab
at the Center for Drug Discovery, Design, and Delivery at Southern
Methodist University has been working to find such drugs to inhibit the
activity of P-gp.
In order to screen compounds for inhibition, much time and money has
been spent on purifying the protein from yeast cells. In response, Collette
Marchessault in the Vogel-Wise lab has proposed a high-throughput
screening method to increase the number of compounds that could be
screened and, in turn, provide for a wider set of data to further the search
for effective inhibitors. I have worked with her this summer to introduce
the human protein P-gp, encoded with the MDR1 gene, into a more
versatile bacterial system in order to increase efficiency and reduce cost.
We have worked to get E. coli to take up the plasmid encoded with the
MDR1 gene so that it can express P-gp. Doing so would allow other
undergraduate and graduate students to screen for potential inhibitors of P-
gp in a more effective and efficient manner.
Methodology and Results
1. Ligation of pet24a-glpF and MDR1 (Fig. 1)
Acknowledgments
I would like to thank Dr. Wise and Dr. Vogel for this wonderful
opportunity and their continuous guidance throughout my time in the lab.
I also would like to thank Collette Marchesseault for taking me under her
wing and letting me contribute to her project. I extend my gratitude to the
rest of the Vogel-Wise lab for teaching me the various lab techniques and
being so welcoming. Finally, I thank Dr. Barbara Fishel from the
Hockaday School for facilitating this research opportunity.
Methodology and Results
2. Transformation of CM2 into BL21 and DH5α, two strains of E. coli
cells using the NEB High Efficiency Transformation Protocol.
3. Plated the cells, Incubated them overnight, and Counted colonies
4. Grew Overnights of the colonies in LB broth
5. Plasmid Preparation [Mini-Prep] on the overnights to isolate
plasmid DNA by means of centrifuging and re-suspending the formed
pellets along with a series of buffers and washes, as indicated by the
Zyppy Plasmid Prep protocol.
6. Polymerase Chain Reaction [PCR] of BL21 and DH5α cells to look
for the 5000bp insert to confirm that MDR1 is in the plasmid. Used
NEB Protocol for PCR Using Q5 High-Fidelity DNA Polymerase
along with T7 and T7term primers, which targeted the DNA region
that included glpF + MDR1 (Fig. 2).
7. DNA Purification: used a series of buffers and centrifuged PCR
products, as indicated by the Zymo DNA Clean and Concentrator Kit,
to isolate and purify DNA.
8. Ran PCR Products on a 0.6% Agarose Gel (Fig. 3)
Conclusions
Based on the sequence data, we can conclude that the E. coli did, indeed,
take up our plasmid with the MDR1 gene. In its entirety, the sequence
highlighted some point mutations in the MDR1, but none of them
impacted which amino acid was encoded and were thus deemed
irrelevant. Furthermore, there were 6 cysteine to alanine mutations, all of
which were accounted for since they were engineered to be there. Two
other mutations were merely natural variants in the gene, and the final
one was a glutamic acid to cysteine mutation, engineered to keep the
protein inactive during this stage of experimentation. The sequence data
thus proved that the MDR1 sequence was complete and correct in the
newly engineered bacterial plasmid, CM2.
Discussion
Since we now know that we have the MDR1 gene in the plasmid, the next
step would include activating the gene so that it expresses the protein P-
gp in the bacterial system. Activating the gene would require a
mutagenesis experiment to mutate the cysteine back to glutamic acid in
order to produce active protein. With P-gp in the bacterial system, high-
throughput screening would be possible and prove to be an efficient and
effective means of finding inhibitors for P-gp.
Furthermore, experimentation has begun to induce the E. coli cells
to produce the glpF-MDR1 fusion protein. In order to do so, the bacteria
has to be grown in the presence of IPTG, a molecule that binds to the
repressor that keeps the glpf-MDR1 sequence from being transcribed.
Once the repressor is removed, the bacteria should be able to produce the
fusion protein. P-gp can then be isolated from the bacteria and used for
further experimentation.
This bacterial system is much more versatile and effective model
system than yeast. Once P-gp is expressed and isolated, research on
potential inhibitors can be conducted in an efficient manner.
Figure 1. Plasmid CM1 shows plasmid pet24a with the addition of the glpF gene
between the two enzyme cut sites. Plasmid CM2 shows the result of the ligation of
pet24a-glpf (CM1) and MDR1.
Figure 2. PCRs use thermal cycling for DNA melting and enzymatic replication of DNA.
The Polymerase enzymatically assembles new DNA strands from nucleotides by using
DNA strands as templates and DNA primers for DNA synthesis. The DNA primers are
complementary to a DNA region that is then targeted for amplification
For further information
Please contact Dr. John Wise ( jwise@smu.edu) at the Southern Methodist University or
visit the website for the Center for Drug Discovery, Design, and Delivery for more
information or to learn more about the ongoing research at SMU.
http://www.smu.edu/Dedman/Academics/InstitutesCenters/CD4
Figure 3. For our gel, we filled the first lane with the 1kb DNA ladder, the next three
with purified DNA from BL21, and the final three with purified DNA from DH5α. Gel
electrophoresis separates DNA fragments by length in order to estimate the DNA size;
all samples were at the 5000bp mark, indicating that MDR1 was in the plasmid.
Furthermore DH5α-10 yielded the brightest band (and therefore had the highest
concentration of DNA), so it was used for the subsequent PCRs.
Methodology and Results
9. Subsequent PCRs on cleaned DH5α-10 resulted in PCR products that
could be used for DNA sequencing. Since the MDR1 gene is almost 4000
nucleotides long, we had to run a series of PCRs in which we used
different forward and reverse primers to target different sections of the
plasmid until we had all of the DNA fragments. For these PCRs, we used
the NEB PCR Protocol for Taq DNA Polymerase with Standard Taq
Buffer.
10. Cleaned the PCR Products with the Zymo DNA Clean and Concentrator
Kit and Ran the products on a 0.6% Agarose Gel. We had estimated the
sizes of the DNA segments earlier based on the location of the primers,
but we ran the gel to confirm the sizes and determine the concentration of
DNA for each segment of the plasmid (Fig. 4).
11. Sequenced the DNA through LoneStar Labs to see if the entire MDR1
gene was in the plasmid (Figs. 5, 6)
Figure 4. Two of the results of
the gel electrophoresis on the
cleaned PCR products of c10 are
shown to the left. “1471” and
“1624” refer to the locations of
the targeted DNA.
Figure 5. Chromatogram data showing the DNA sequence of a portion of the
MDR1 gene. Each color correlates to a different nucleotide, labelled at the top of
the graph: Adenine, Guanine, Cytosine or Thymine.
Figure 6. The sequence data (portion shown above) matched up with the genetic
sequence of MDR1 and thus allowed us to conclude that MDR1 was, indeed, in
our pet24a-glpF-MDR1 plasmid.