Taking time out from molecular biology to attend a fund raiser.

We met after her second day in the Targeted Genome Editing Workshop when she learned the intricacies of working with Zinc Finger Nucleases to create knockouts, gene knockins, and integration of a selected gene of interest into a cell line.

Dr. Chilton is a two-time graduate of Biouniversity workshops.  Her first workshop focused on using RNAi technology, with the goal of knocking down difficult genes in her rabbit model.   During the RNAi workshop she learned that Sigma was unveiling a  technology for gene knockout,  knockins and/or Integration into a cell line.   Dr. Chilton nearly tackled the speaker to get more information! She intends to apply knockouts and knockdown to human cell lines.

What did the RNAi workshop do for you?

The workshop showed me the “La Brea Tar Pits” (or “sticky wickets” if you’re British), to look out for in using the technologies.  You get to work with the people who developed the technology, and really learn the in’s and out’s.  To me it’s an invaluable service.

What made you want to come back for a second workshop?

What the Sigma team tries to achieve is very unusual. The spirit in this [Workshop] room is one of understanding and cooperation.  It’s like you are saying “Yes, we make products, and we want them to work in your lab.”  You want to sell me one good kit that will work and WILL make my science go forward.

To me this feels as genuine as it can possibly feel.  Ultimately the partnership and personal commitment make the difference in using these new technologies.

Do you plan to use ZFN Knockouts and RNAi together?

Yes.  I plan to eventually use them together.  I want to remove our transcription factor (RUSH, a protein highly conserved between rabbit and human, with the human protein named HLTF) then knockdown its  interactors Egr-1 and c-Rel using shRNA

Your shRNA didn’t work after your first workshop. What happened?

I was trying to apply human tools to a rabbit model, and it’s quite frustrating.  The rabbit model does a wonderful job of expressing my genes, but the tools that are available aren’t always applicable to the model.

How about the ZFN’s?  How are they working?

I’m looking forward to using ZFNs in a human model.  ZFNs provide much greater versatility and have allowed researchers to knockout 3 genes in the same cell line, you just can’t do that with knockdown technologies like siRNA and shRNA.  We are working on the RUSH protein and we’ve identified the Jak2/RUSH pathway for prolactin signal transduction in rabbit uterus.  The work has been very well received by the scientific community. Now we plan to do more in a human model!

Where did your bio begin?

It started when I was very young.  When I was a child I wanted a microscope for my 9th or 10th birthday.  Then I asked my dad if he couldn’t cut himself shaving so I could have the blood to look at under my microscope…he wasn’t pleased with that!

The real trick for me was finding out what kind of biology I wanted to do.  I have 3 degrees in Zoology.  Ultimately I discovered that Molecular Biology is very intellectually satisfying.

I have loved science since the beginning, and I love it more today than I did the day I started!

Learn more about Sigma Life Sciences workshops, and find a schedule of upcoming events please visit  sigma.com/workshops.

The strategy combines high-throughput generation of mono-specific antibodies with protein profiling in human tissues and cells using immunohistochemistry (IHC) on cell and tissue microarrays (TMAs).  Dr. Ponten covers the development and validation of Prestige Antibodies® powered by Atlas and explores the use of these antibodies in the search and characterization of biomarkers related to cancer.

The webinar concludes with a brief overview of Your Favorite Gene Powered by Ingenuity given by yours truly.
I also recommend Kyle’s video if you want the highlights of this powerful research tool.

Speaker:  Dr. Fredrik Ponten, Professor, Uppsala University and Vice-program Director, Human Protein Atlas Project

With Locked Nucleic Acids (LNA^®), you CAN use a shorter probe in your assay and expect greater sensitivity and discrimination for your gene target.

Dr. Ashley Heath of the Sigma Custom Products Technical Service team tells us below about the details of this novel alternative to standard DNA probe chemistries.

What are locked nucleic acids (LNA®)?

LNA® is a novel type of nucleic acid analog that contains a 2’-O-4’-C methylene bridge.

This oligo modification

• Confers greater thermal stability
• Reduces its flexibility
• Increases hybridization interactions of the base.

A comparison of LNA® to DNA is shown below.

I use dual-labeled fluorescent probes (DLFPs).  What’s the difference?

DLFPs contain DNA monomers and are generally 25-35 bases in length. LNA® probes contain a mixture of DNA and LNA® monomers and are generally 14-17 bases in length.

Do I need to alter my protocol to use LNA® based probes?

No.  LNA® probes are hydrolysis probes like DLFPs. Experimental set up and data collection are the same.

What are there advantages to using LNAs®?

LNA® probes have a greater thermal stability compared to conventional DNA or RNA.  Additionally it is possible, with strategic placement of the LNA® bases, to optimize the Tm level and hybridization specificity.

Incorporating LNA® chemistry into your qPCR probes can improve performance in SNP discrimination, multiplex assays and when working with problematic target sequences.

Read the full list of LNA® FAQs

Dr. Heath and his fellow technical service team are available to answer your toughest questions AND to provide FREE qPCR primer and probe design.  The team uses Beacon Designer to routinely provide design for primers and a variety of probes including dual labeled probes, LNA® probes, Molecular Beacons, Scorpions™ and LightCycler® probes.  Now that’s service!

Experienced technical support is just a phone call away or visit sigma.com/designmyprobe to submit your design request.

Dr. Heath (Senior Technical Service Scientist) has extensive experience both in academic research and in the life sciences industry. He holds a D.Phil. degree from the University of Sussex, England, in Physiology and Anatomy, and has continued education in Infectious Diseases from the London School of Hygiene and Tropical Medicine. Prior to joining SIGMA in 1996, he held faculty positions at the Pennsylvania College of Optometry, Philadelphia, PA. and at Baylor College of Medicine, Houston, TX. At SIGMA he has worked in the areas of R&D, technical sales and support, and business development.

**LNA® oligonucleotides produced under license from Exiqon A/S.

HPA publishes the Immunohistochemistry data profiled across normal tissue, cancer cells and cell lines for free to everyone.

IHC image in normal spleen for Anti-ADCY10: Cat. No. HPA015243

Then Sigma Life Sciences works with Atlas Antibodies to make Prestige Antibodies^® available to everyone.

On YFG you can search for a protein, gene, or other molecule, by pathway, function, disease, etc. etc.
Here we’ll use Epidermal Growth Factor Receptor (EGFR)
You see to the right all the data that’s available. To find the HPA antibodies with buckets of data, click “Antibodies”.

Here we have many many  antibodies associated with EGFR.  Then, thanks to HPA and Atlas, we have a select group of antibodies tested for activity in normal and cancer tissues.

Find these on the Product page designated by a Product # starting with the letters “HPA”

Click on it…and you have IHC images from HPA showing the binding activity of  Antibody HPA018530 .

If you need to review the normal tissue expression across the board or the data on cancer cell lines and tissues…click on the link “Human Protein Atlas characterization data”

Dig into the data here.

Enjoy your fun IHC data treats.  We surely do.

Keep looking and you will also find some ImmunoFluorescent data, and Western Blots.

Of course, you may have reasons why this kit isn’t for you so we think it is time for a top 5 list, our first on SigmaBioBlogs.

Reasons NOT to use the CompoZr Targeted Integration Kit:
1. You aren’t in a hurry to publish your results.
We are talking about the possibility of getting rapid biallelic insertion through a single transfection in as little as 48 hours….

2. You don’t mind inefficiency.
Genome editing using homologous recombination has served the research community well for the past two decades but it is highly inefficient.  A one in a million event, this method also requires sophisticated selection strategies.  The well-validated ZFNs within the CompoZr Targeted Integration Kit cut the DNA with high efficiency at the AAVS1 site.  The success rate is up to 3 to 4 orders of magnitude higher than spontaneous homologous recombination events.

3. You want varied expression levels and stability.
Previous methods to create transgenic cell lines use random integration of a plasmid.  The result is greatly varied expression levels and expression stability.  Use of a single genomic locus means stable, uniform expression levels.  We chose the AAVS1 site, a safe harbor locus that is ubiquitously expressed and its disruption leads to no adverse effects on the cell.

4. You like complexity.
We all know people like this and you may be one of them.  You can’t help but give a dissertation when a simple Yes/No answer would have sufficed.  If this is you, then this kit is just too simple for your taste.  Gene integration is at a single endogenous locus. The need to engineer cell lines with transgene landing pads is eliminated.  I mean, come on, that was the fun part, right?

5. You want us to design a custom ZFN!
We didn’t believe your other excuses but this is a reason we can rally behind.  We can rapidly design, assemble and validate a ZFN pair targeting YOUR gene of interest.

Now that we’ve established that reasons 1-4 don’t apply in your case, let’s go back to #1.  You DO want to be the first to discovery (this is what we love about you).  Did you know that the recently released Targeted Integration Kit could have been in your hands a year ago?  Participants in our Validation Testing Program obtain early access to some of our coolest products!

Read what one tester had to say about the program:
I was impressed by the validation testing program for a number of reasons. I had frequent and prolonged phone and email conversations with multiple scientists at Sigma and they listened intently to my input. Further, they went back and redesigned the kit a few times, trying to address where we (and others) had problems. It was clear to me that Sigma very stringently tested this kit to make sure that it will work as advertised.

Researchers who tested the CompoZr Targeted Integration Kit conducted our desired protocol using just 1/5 of the kit!  That is a lot of remaining product for your research.  Also, rapid responders receive incentives (such as gift cards) in addition to free product.  Find out more!

Sarah grew up a farm girl in Western Kentucky.  She always loved nature, climbing trees and pitching in to help run the family farm (Here’s where our stories diverge..  I did the cooking, fed the family and the cuter animals)

While in college she met Joe Kuc, who was studying signal transduction in plants. Impressed with his approach, she adopted some of his philosophies into her own teaching style.  This includes the allocation of her time as follows:

1. 20% Outreach to the community
2. 40% Research
3. 40% Teaching

After receiving her PhD, she did a post-doc with with NASA researchers at  NC State as part of NSCORT The NASA Specialized Center of Research and Training. There she studied the effect of gravity on plant growth, and has been studying it ever since. For Sarah, the real fascination lies with the fact that  plant life has intelligence independent of a nervous system used to relay external signals from the environment..

She believes that plants might be the highest life form on earth…if you challenge that point she will let you know about totipotency…the plant cell’s ability to differentiate…then undifferentiate, assuming an entirely new role.

This post brought to you by Sigma Life Science Plant biotech products

Proteomic Peril was inspired by Natalie’s time waiting for her husband at the ASMS (American Society for Mass Spectrometry) meeting a couple of years ago.  Her husband, Life Sciences veteran Aaron Sin (Sigma’s Business Development person for Proteomics) begged to differ.  That year he learned about applying micro-fluidics in the field of proteomics.

Well we are happily attending the  58^th ASMS Conference on Mass Spectrometry and Allied Topics again this year, and are bringing information on ways to use AQUA Peptides to do protein quantification, SEPPRO depletion columns to find low abundance proteins, Universal Proteomics Standards for Mass Spectrometry, and also Supelco analytical will be available to talk HPLC columns, LC-MS and much more.

Sigma Life Science is taking part in the following presentations while at ASMS, we hope to see you there.

Poster #502– Tues May 25, 10:30am-2:30pm

Comparison of Fused Core and Porous Particles for Fast LC-MS-MS Analysis Using Conventional LC Instrumentation

Oral Presentation  #1124  Tuesday May 25, 3:10pm Hall 3

Dealing With Phospholipids In Regulated Bioanalysis

Workshop – Tuesday May 25, 5:45-7pm

LC-MS-MS Analysis of Biomarkers and their Impact on Drug Development–Organized by Pharmaceuticals Interest Group

If you are asking the question “What are zinc finger nucleases?” then you are in luck!   Sigma Life Science has created a series of FREE, live online seminars to introduce you to the technology and to delve into specific applications of zinc finger nucleases.

If you missed the introductory webinar held on May 6, the recorded webinar Introduction to Zinc Finger Nuclease Technology will be available for you to experience at your convenience.  We spent 30 minutes listening and this is just a taste of what we learned.

1.  What are ZFNs?
ZFNs are engineered DNA-binding proteins that act as “genomic scissors” to create a highly targeted double strand break in genomic DNA at any specified locus. The double strand break is repaired by the cell, leading to gene knockout or gene modification only at the intended locus. This technology will knock your genes right off!

2.  Design consideration
A two pronged approach of bioinformatics and protein engineering maximizes the specificity of ZFNs. A sophisticated algorithm is used to look for unique sites within the genome (target sequences that do not have homologous sequences even with 1-4 base pair mismatches) and avoid regions such as repeat elements, SNPs and splice variations. The Fok I nuclease is also engineered to function only when two different zinc finger nucleases come together, increasing specificity. Nothing like a clean break!

3.  Using ZFNs
Dr. Collingwood will cover the various applications of Zinc finger nucleases for targeted gene knockout and target integration.  The overall advantages for using zinc finger nuclease technology in both approaches include: modification of endogenous loci, ZFNs expressed transiently, no selection required (it is that specific!) and monoallelic or multi-allelic disruption/insertion.

Are you ready for more advanced topics in Zinc Finger Nuclease Applications? Of course you are.

May 27, 2010 - Gene Knockout in Cell Lines
Dr. Collingwood provides examples of both single and multiple gene knockouts drawing from work with transformed cell lines, primary human cells and multi-potent stem cells.
9 AM Central Time
4 PM Central Time

June 15, 2010 – Targeted Gene Integration
Join Dr. Collingwood for a discussion of various applications of gene integration including gene tagging (such as infusing GFP directly to the endogenous gene) and modification at the endogenous locus to recreate or correct disease-related mutations.
9 AM Central Time
4 PM Central Time

Register to attend the live webinars or check back at sigma.com/zfnwebinar for the recording to be posted.  We hope to have the recording of the first webinar posted soon.  Until then, check out this video on ZFNs.

CompoZr ZFN Technology for Targeted Genome Editing

We are looking forward to hosting another successful event that pulls together researchers from across Mexico from both the Academic and Industrial sectors.

The focus is on understanding the development, uses and applications of bioactive small molecules crossing the disciplines of chemistry, bionology, and biotechnology.

Register now!

2nd Symposium Convestav/Sigma-Aldrich: Bioactive molecules: The frontier of chemistry and biology

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Dr Gerardo Corzo Institute of Biotechnology, UNAM

Potential of peptides from spider venom as therapeutic agents

Dr Adela Rodríguez Instituto de Química, UNAM

Structure-immunogenicity of natural and recombinant forms of the allergenic proteins of natural rubber.

Dr Benjamin velasco (CONADE)

Determination of bioactive substances in high performance sport

Dr Daniel Martinez Fong (CINVESTAV)

The Polyplex Neurotensin: New drug for Parkinson’s disease and cancer.

Dr Liliana Quintanar (CINVESTAV)

Study of the interaction of copper with amyloidogenic protein: from mad cow disease to Parkinson’s

Dr. Lorenza Gonzalez-Mariscal (CINVESTAV)

Use of peptides for analysis of the role of tight junction molecules

Dr Ricardo Robledo (National Public Health Institute)

Importance of measurement of biochemical indicators in the Health and Nutrition Studies

Dr. Shahriar Mobashery (Notre Dame University)

Small Molecules and the Bacterial Cell Wall

This post brought to you by the Sigma Life Science Biomolecules Team

• Bioactive small molecules

On Earth Day, we posed the question to our Facebook fan page, “What are you doing to support the environment?”, and found responses were unanimous! Recycle! RECYCLE!  Aluminum, paper, buffer bottles, empty pipette tip boxes, cardboard boxes.  We even heard about one lab that is doing the ever popular Green Chemistry experiments.

Recycling and pipette tips
Reducing freezers used, and energy spent maintaining samples. - Green (Energy efficient) labs (from Biocompare)

And here, a study at the NCI is sited for a successful recycling program.

So, Dr. Kelrick, molecular biologists may not be able to recycle pipette tips (yuk…and yet), but they are working to minimize the impact of their human bio on your eco-bio.