Hello and welcome to this VectorBuilder presentation which I've titled “How To Design A Good Vector for Your Cancer Research." First of all I'd like to thank you all for being
present here today and I want to thank the LSE organizers for putting this conference together. My name is Joan Marcano and I am the US/Northwest territory manager for
Vectorbuilder. I want to remind you that this presentation is recorded but I will be available at the end of it to answer any questions that you might have. In today's talk I will highlight
a case study which uses VectorBuilders Vector Construction Service to achieve their goal of understanding tumorigenesis in a particular cell line. Before I do that I wanted to go over
other stuff that you can do with vectors. The main thing that people can do with vectors is expression analysis. By this I mean the gain or loss of a function of a particular gene that
you might think is involved in a tumorigenesis pathway, for example. Other things that you can do is direct gene modifications in which you edit the genes throughout mutations.
You can add tags or fuchsia to a fluorescent coating. After that you can use screening of libraries in which you for example use a CRISPR library to target different genes in a pathway
and try to parse out how they interact with each other. You can also look at those those
genes are regulated by targeting the intergenetic regions or non-coding regions and look
at the transcriptional activation or repression of those genes. Finally, you can also actually
monitor either the DNA/RNA or the protein inside the cell by designing specific vectors that
have different proteins or nucleic acid for visualization in the cell. The case study that I'm going to present to you today comes from work that was published from the Trent lab at
The University of Miami. The goal of this research was to analyze the role of IDH1 mutations in a chondrosarcoma tumorigenesis by knocking out that IDH1 mutant.
To give you a little background, the IDH1 or isocitrate dehydrogenase enzyme converts isocitrate into alpha-ketoglutarate as part of the normal TCA cycle. Instead of making
alpha-ketoglutarate it makes 2 hydroxyglutarate also known as 2-HG. 2-HG is a known oncometabolite which inhibits different enzymes including demethylase. It is thought that
now it leads to a hypermethylated state of DNA which results in the activation of oncogenes and the inactivation of tumor suppression genes. To help them with their research
VectorBuilder constructed two lentiviral plasmids for the Trent lab. The first plasmid contained the wild-type IDH1 mutant to be expressed under the knockout cells and
a GFP marker. The second one contained ONLY the GFP marker and was used as a control. You can see here in the right pane that both the plasmid containing the
wild-type IDH1 and the control vector both transduced efficiently both of their cell-lines containing the IDH1 knockouts. After transducing their cell-lines, the group then
looked at the alpha-ketoglutarate levels and the 2 hydroxyglutarate (2-HG) levels. In this left pane measuring the alpha-ketoglutarate (a-KG) levels we can see that the parental strain
containing the mutant strain produced lower levels of a-KG then the mock which carries the wild-type version of the a-KG. In the knockout cell-line in both cases
we can see that the a-KG levels starts to rise up but then the IDH1 mutant is expressed in the contents of that knockout we see even higher levels of a-KG down the
parental line. The most important thing about this study is that once we look at the 2-HG levels we see that overexpressing the wild-type IDH1 enzyme in the
knockout cell lines did not increase the 2-HG levels, confirming the evidence that the wild-type IDH1 enzyme does not produce 2-HG. With this new evidence the group
was able to directly show in cells the role of the IDH1 mutant in tumorigenesis and was able to map out the signaling mechanism by using Vectorbuilder's lentiviral system.
So, that was a great example of how we were able to help the Trent lab but the real question we want to answer is how can Vectorbuilder help your research?
Vectorbuilder can be your research partner and your one stop solution for all your vector and virus needs. To achieve that we provide you first with an online Vector Design studio
which you can use for free and allow you to easily design all the vectors you need and we can even give you advice on them.  We don't only help with design but we can actually
construct the vector. We do the cloning for you. If your vector is for viral delivery we can package that vector and then deliver your titer guarantee viral particles to be used in
your experiment. You might need more than one vector. You might need a library. We can construct and advise you ont hat as well. We do BAC Recombineering and we can
take it all the way as to produce a stable cell-line for you. What makes our vector construction service unique? First of all, will be the variety of systems that are already
standardized in our Vector Design Studio. These are sorted by applications that means gene expressions, shRNA, CRISPR and so on. Not only do we have a variety of vectors
for different applications but these vectors are also standardized for other model organisms beyond mammalian cells such as zebrafish, drosophila, and yeast.
Another thing that we have standardized are the vector components that you can use and choose to customize your vector. By that I mean, the promoters, the markers, the
linkers, and all I will talk a little bit more about a couple of those elements in a little bit. We can construct these vectors at an unbeatable price and turnaround time.  We will
100 % guarantee all the vectors that we construct for you. We will sequence them before sending it to you and they are guaranteed as I said.
Now the questions that we might ask now is, what makes a good vector for your experiment? There are many different things that you should take into consideration
when doing that but today I will be just going over a couple main things that you should keep in mind when choosing which vector you're going to use. The first thing that you need
before designing your vector for your experiment is what is the plasmid background that you want to use? Now these are the most popular plasmid backbones that
we use to construct vectors for our customers and each one of them has several advantages or disadvantages. You will have to choose carefully based on what cells are you going
transfect and what you want to achieve. Most notable are the Lentivirus and AAV's. These two are viral vectors and the main difference between these two is that lentivirus is a type of
retrovirus in which the plasmid will enter the cells and actually integrate into the genome of the host. This allows you to do stable expressions or make stable-cell lines with gene
They have a moderate cargo sizing which lets you input several genes up to 9.2 kilobases(kb). AAVs on the other hand will give you transient expression. This means that when your
plasmid is delivered to the cells it will not integrate into the genome of the cell. AAV's a smaller cargo space so you can't input as many genes as you can into the lentivirus
but the AAV's can be packaged with different serotypes which allow for specifc infection of different tissues. These two (Lentivirus and AAV) are the most popular viral vectors.
The other main one is just the regular more traditional regular expression vectors. That also give you transient expression but they have very, very large cargo space where
you can input many genes, up to 30 kilobases (KB). Our team can advise you based on the cell type that you're going to use. We can help you based on our experience
or based on publications on what method you should use to transfect your cells. Once you have chosen what plasmid backbones you want to use, you can start
thinking about what promoter you're going to use. To remind you, the promoter is the sequence element that drives the expression of your gene or sequence of interest.
You can achieve different levels of expression using different promoters. We and others have characterized the expression of different promoters and different cell types.
We can definitely give you advice on what promoter to use depending on the cell type that you're planning to transfect and what level of expression that you want to achieve.
These promoters that are here are what we know as constitutive promoters. They have been shown to express in a variety of different cell-types. However, I wanted to add
Vectorbuilder also has standardized a variety of tissue-specific promoters, meaning that these promoters are regulated in a way that they will only express the gene of
interest in a particular tissue. This allows you to do selective expression, particularly when you're doing viral infections. Sometimes you want to express multiple genes from
the same promoter. There are two main ways you can achieve that. The first one is by the use of Internal Ribosomal Entry Sites (IRES) in which you separate
your genes by this viral sequence. This provides another entry for the ribosome to translate your second gene of interest as a separate protein. We've seen that IRES have different
efficiencies and different cell types. This data that we can definitely use to advise you if it's reasonable to use an IRES to separate your genes. Or if you will see enough
expression of your second gene based on the cell-type that you want to use. The other main way that you can express two genes from the promoter is by separating 2A viral
linkers. The way these viral linkers work is that your two proteins are expressed as a single polypeptide but then there is a cleaved reaction that will separate
these two proteins into individual proteins. Now there are different viral 2A sequences and they can cleave the polypeptide to different efficiencies.
Just note that when you use these 2A viral linkers you will add a couple extra amino acids at the C terminus of your first gene and one amino acid occurring into the
end terminus of your second gene. Sometimes for functional experiments this is not acceptable. On other occasions this is fine. These are very popular ways to express
two genes from the same promoter. The other alternative is that you can express two different genes from separate promoters. However the arrangement of these
vector components do matter in terms of how much expression you're going to see from different elements. This is very evident in viral plasmids. Here are some examples where
we have mCherry and EGFP expressed from the EF1A promoter or the CMV promoter. In the first page we have mCherry being expressed first and EGFP being expressed
second. Now when you look at the EGFP channel compared to the mCherry you can see that the mCherry is expressant to a higher level. Now if we switch the order of the genes
in these plasmids and have the EF1A promoter expressing the EGFP, you can see from the fluorescent images that now the EGFP is expressed at a higher level than the mCherry.
This sort of data highlights the things that we have learned. Not only from internal data but from the literature. We can use this to advise you on how to construct your plasmids
so you can have the expected results from that vector. The last thing that we need to choose is what marker you would like to use for your experiment. This could be
either fluorescent proteins or antibiotic resistant gene. On our website, you can look at all the different fluorescent proteins that we have standardized and look at the
emission and excitation of these proteins to help you make a better decision on what sort of fluorescent proteins you want to use for your experiment.
Again, we can help you choose if you have any questions or if you're unsure. Some people for example want to use two colors in their experiment and we can help
you or make recommendations on what two colors would work best. Either based on the equipment that you have or to choose different fluorescent proteins that will not
interfere with each other. We can help you with two-color or three-color experiments. Also if you want to use a fluorescent resonant energy transfer in your experiment we can
tell you which will work at FRET partners. There are a lot of things that you would need to think about but we are here to help you to make the best decisions for your
experimental needs. The other service that I want to highlight today is our Viral Packaging service. We can package a variety of viruses, including Lentivirus, AAV's,
Adenovirus, MMLVs, MSCV, and Baculovirus. For all of this we can achieve different titers. This table highlights a little bit more details of each of the services. As a note, we
can make these viruses for cell culture applications or we can ultra purify them so you can use them to inject for animal experiments. The standard QC includes the
titration of the virus will show the transforming units or genome copies per mil. This will help you achieve more consistency in your experiments and better planning for the future.
You can get the same concentration consistently from the preparations. We will test for bacteria, fungi, and mycoplasma. We can also do the standard test as well as some
additional services upon request. We have expanded our capabilities in viral packaging services substantially within the past few years. We are very proud of the service so
please let us know if you have any needs or if you have any questions regarding this and we can help you out in choosing the best service and the best quantities that you need
for your experiment. Today I am not going to go any deeper on the other services that we provide. I just wanted to quickly give you some highlights from our custom vector cloning
and virus packaging services. Please do note that we can provide advice and help construct your library of plasmids. We do BAC Recombineering and Stable Cell Line
Generation so we can really be your research partner at different levels and stages of your research. So, one quick note that I wanted to make is that we are also taking this
expertise of vector construction and viral packaging and offering GMP Manufacturing to meet major GMP regulation guidelines in the US, the European Union, Japan, and China.
We are ready to provide this service so if you are thinking about any clinical trials using DNA or viral therapies, please ask us about this service and we will be happy to
give you more details. With that, I would like to thank you for your attention and give a shout out to our research partners in the industry and academic laboratories. Thank
you to the LSE organization for putting together this meeting and please visit our virtual booth. I'll take any questions now. Thanks again!
