Cancer formation is a heterogeneous and complex process, involving many factors and cellular signaling pathways. There are more than 1,200 potential cancer biomarkers identified in the literature by a 2006 review, which, if analyzed in multiplexicity, may provide the best potential for reliable and early detection of cancer. Many proteins including most cancer antigens become post-translationally modified (PTM) during the “secretory process”, which involves of a journey from their site of synthesis in the rough endoplasmic reticulum (ER), through the Golgi apparatus and then to various cellular and extracellular destinations.
Examples of protein modifications include glycosylation, phosphorylation, acetylation, and amidation. Of these, the most complex procedure is glycosylation, involving several enzymes. There are increasing demands for these glycosylated human proteins in good quantity, purity and affordability by the scientific community to perform fundamental and clinical studies in relation to cancer. Such proteins can not be expressed in bacteria or yeast because those cells do not carry out equivalent PTM as in mammalian cells. Allele Biotech has chosen a modified baculovirus expression systems (MBVES) as the main method for producing glycoproteins and the proposal was chosen by the NCI for funding of $150,000, approximately 75% of the cost of producing 10 glycosylated cancer antigen proteins in the first phase of 6 months. The remaining funding of ~$50,000 mostly in indirect costs will be covered by Allele’s own funds. This SBIR contract will be partially subcontracted to the University of California , San Diego (UCSD) during the glycan analysis phase. The work will be performed in Allele’s San Diego facility and UCSD’s GlycoTechnology Core facility.
This news is released by Allele Biotechnology & Pharmaceuticals, Inc. through AlleleNews, for in-between experiments
Monday, September 28, 2009
Saturday, September 26, 2009
The economy recession is most likely over, says who?
The economy recession is most likely over, or so says the federal reserve chairman Ben Bernanke. Do you feel it? Are you seeing increased job opportunities when you leave your current lab or security if you have a post-postdoc position? In our industry, where the health of the economy is mostly measured by research budgets of individual labs or research groups, occasionally by budgets for contracting or licensing fees, the change, if any, is still hard-to-find. But hiring at academic institutes like UCSD seems to have picked up lately, probably due to addition grants from the Obama administration’s stimulus programs. At the same time, individual NIH R1 grants have been creeping up to easily around 1 million a year, program grants 3-5 millions. With more stimulus money kicking in to academic labs this fall, it is expected that the situation will further improve. Comments welcome.
Notes about recent jobs in Pharma/Biotech: since our last blog about massive Pfizer layoff of scientists in 02-09-09, a major layoff in the big pharma sector came from Merck, which announced on 06-11-09 that it would cut 16,000 jobs after completing its merger with Shering Plough. On 09-14-09, Eli Lilly reported job cots of 5,500 or roughly 14% of its work force. There are areas in the country where people report about the economy as “I went to 2 grocery stores and 3 discount department stores over one weekend, and you could do cannon shooting practice in there without hitting a person.” Again comments welcome here, if you believe in a turnaround, or it is all doom and gloom to you. Btw, the History channel has been cranking up the 2012 theories for a couple of months now, if you like the doom and gloom theories.
Note added in proof: As reported in Science yesterday, “a new analysis of the grantsmaking process at the National Institutes of Health (NIH) lifts the veil on how many grant proposals are funded even though they fall below a cutoff based on peer-review scores…at least 19% of NIH’s basic research portfolio is funded for reasons that go beyond quality.”
Notes about recent jobs in Pharma/Biotech: since our last blog about massive Pfizer layoff of scientists in 02-09-09, a major layoff in the big pharma sector came from Merck, which announced on 06-11-09 that it would cut 16,000 jobs after completing its merger with Shering Plough. On 09-14-09, Eli Lilly reported job cots of 5,500 or roughly 14% of its work force. There are areas in the country where people report about the economy as “I went to 2 grocery stores and 3 discount department stores over one weekend, and you could do cannon shooting practice in there without hitting a person.” Again comments welcome here, if you believe in a turnaround, or it is all doom and gloom to you. Btw, the History channel has been cranking up the 2012 theories for a couple of months now, if you like the doom and gloom theories.
Note added in proof: As reported in Science yesterday, “a new analysis of the grantsmaking process at the National Institutes of Health (NIH) lifts the veil on how many grant proposals are funded even though they fall below a cutoff based on peer-review scores…at least 19% of NIH’s basic research portfolio is funded for reasons that go beyond quality.”
Thursday, September 24, 2009
Retroviral Vectors with Integrated oriP/EBNA1 for IPSC
The new product of the week of Sep 21-27 is the retrovirus plasmid sets that contain a built-in episomal expression system. As we have discussed previously, OriP/EBNA1 system originated from Epstein-Bar virus, which allows the establishment of stable episomes at 5-20 copies per cell, and duplication once per cell division.
By using the oriP/EBNA1 episomal system, reprogramming cDNAs can be expressed at prolonged time period in reference to plasmid transfection, without integration into chromosomal DNA. A paper published in PLoS One on Sep 18, 2009 by Marchetto et al. showed that by using such a system (on different plasmids) the authors were able to create induced pluripotent stems cells (iPS cells,) effectively from human embryo neural precursor cells.
The Allele pCHAC-EBNA system has dual functions: it can be ready-to-use plasmids for episomal expression of Oct4, Sox2, c-Myc, Klf4, or Nanog and Lin28 by a simple transfection into target cells; it can also be packaged into retroviruses by transfecting into the Allele Phoenix Retrovirus packaging Eco or Ampho cells. This product group is officially launched today. It should become a highly convenient and unique tool for iPSC-related studies.
By using the oriP/EBNA1 episomal system, reprogramming cDNAs can be expressed at prolonged time period in reference to plasmid transfection, without integration into chromosomal DNA. A paper published in PLoS One on Sep 18, 2009 by Marchetto et al. showed that by using such a system (on different plasmids) the authors were able to create induced pluripotent stems cells (iPS cells,) effectively from human embryo neural precursor cells.
The Allele pCHAC-EBNA system has dual functions: it can be ready-to-use plasmids for episomal expression of Oct4, Sox2, c-Myc, Klf4, or Nanog and Lin28 by a simple transfection into target cells; it can also be packaged into retroviruses by transfecting into the Allele Phoenix Retrovirus packaging Eco or Ampho cells. This product group is officially launched today. It should become a highly convenient and unique tool for iPSC-related studies.
Monday, September 21, 2009
Difference between hESCs and NSC-Derived Integration-Free iPSCs
By introducing Oct4 and Nanog into human fetal neural progenitor cells, the Muotri lab at UCSD, in collaboration with colleagues from the Yeo and Gage labs of UCSD and Salk, was able to create induced pluripotent stem cells (iPS cells) using the oriP/EBNA1 episomal system to introduce the aforementioned Oct4 and Nanog. While it has been shown by others that with less differentiated cells, such as progenitor cells, it is relatively easy to reprogram using two, or sometimes even one, of the commonly used iPSC generating factors (Oct4, Sox2, Klf4, c-Myc, Nanog, or Lin28) as reported in the AlleleNews article “iPSC generated by using a single reprogramming factor” on August 3rd, 2009, the new publication in PLoS ONE analyzed in detail the differences between embryonic stem cells (ES cells) and iPS cells.
There were three groups of genes that changed significantly between hESC versus iPSC and iPSC versus NSC; genes that are important in early embryonic fate including iPSC-expressed factors that are not sufficiently repressed and genes that are upregulated in iPSCs but are silenced in both NSCs and hESCs which may be downstream to the reprogramming genes during dedifferentiation. Their conclusion is that iPSCs may retain the gene expression signature of donor cells in human reprogrammed cells, even with integration-free gene transfer vehicles such as oriP/EBNA1 element containing plasmids. It should be pointed out that this episomal vector system has been previously reported for iPS cell generation by the Thomson group, see:
(http://allelebiotech.com/blogs/2009/03/episomal-expression-of-ips-inducing-genes-no-trace-of-transgenes-afterwards/). In this paper it was also shown, not unexpectedly, that myc-immortalized cell lines can be efficiently reprogrammed.
Marchetto et al. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007076
Note from Allele: Congratulations to Maria, Gene, Alysson and the Gage lab.
There were three groups of genes that changed significantly between hESC versus iPSC and iPSC versus NSC; genes that are important in early embryonic fate including iPSC-expressed factors that are not sufficiently repressed and genes that are upregulated in iPSCs but are silenced in both NSCs and hESCs which may be downstream to the reprogramming genes during dedifferentiation. Their conclusion is that iPSCs may retain the gene expression signature of donor cells in human reprogrammed cells, even with integration-free gene transfer vehicles such as oriP/EBNA1 element containing plasmids. It should be pointed out that this episomal vector system has been previously reported for iPS cell generation by the Thomson group, see:
(http://allelebiotech.com/blogs/2009/03/episomal-expression-of-ips-inducing-genes-no-trace-of-transgenes-afterwards/). In this paper it was also shown, not unexpectedly, that myc-immortalized cell lines can be efficiently reprogrammed.
Marchetto et al. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007076
Note from Allele: Congratulations to Maria, Gene, Alysson and the Gage lab.
Friday, September 18, 2009
Effective Concentrations and Effectiveness of siRNA
RNA oligo is significantly more difficult to synthesize than DNA oligos, mainly because the efficiency of coupling each new ribonucleotide during RNA synthesis is a few fold lower than deoxyribonucleotide during DNA synthesis. Typically, there is an ~10% chance a DNA oligo of 21 bases will have a mutation (most frequently a deletion mutation); for an RNA oligo of 21 bases, as in an siRNA pair, such chance is much higher. Furthermore, after combining the sense and antisense siRNA strands, some RNA molecules will remain as single-stranded thereby not fitting for the RNAi apparatus.
RNA interference is a dose-sensitive process — specificity of gene silencing is meaningful only relative to the active concentration of siRNA used. When the concentration is too low, even the most effective siRNAs would fail to cause gene expression knockdown; when too high, non-specific effects will be duly observed. Therefore, it is essential that the concentrations of siRNAs are measured correctly. When doing so, one must consider not only what the apparent concentrations are by OD260 reading, but also whether the RNA strands are of full-length and whether only dsRNA molecules are counted. This issue might not affect data interpretation if appropriate controls are included in one set of RNAi experiments, but it could have significant influence on conclusions if data from different experiment sets or labs are compared or combined.
HPLC purification currently provides the best means to remove RNA molecules with deletions or remain single-stranded, however, the price tag added by most reagent providers for such treatment has been prohibiting because manufacturers either need to start synthesis at a much bigger scale to obtain promised amount, or they do not promise the delivery quantity at all. The phosphoramidites (oligo building blocks) for RNA synthesis can be 10 times or more expensive than for DNA. Some companies offer alternative purification methods such as a cartridge type device, but they can only remove salt and small impurities, not RNA oligos of shorter lengths accumulated at each cycle of amide coupling. The AllHPLC siRNAs within Allele’s RNAi product line, pre-validated or custom made, are uniformly HPLC purified with 5 OD or 12.5 nmol of double-stranded, annealed siRNA delivered. Allele passes to customers the cost savings from manufacturing our own RNA amidites and other reagents for oligo synthesis. The pre-validated HPLC purified double-stranded siRNA is offered today at $149/12.5 nmol.
Before purchasing siRNAs, even at a low cost of $29 per pair of HPLC purified control siRNA from Allele, researchers still need to consider how well their cells can be transfected. For hard-to-transfect cells, lentiviral vectors carrying a shRNA expressing cassette is often a better choice. To establish stable cell lines, plasmid vectors should be considered. For low cost target screening, the PCR format linear siRNA expression cassettes have advantages.
RNA interference is a dose-sensitive process — specificity of gene silencing is meaningful only relative to the active concentration of siRNA used. When the concentration is too low, even the most effective siRNAs would fail to cause gene expression knockdown; when too high, non-specific effects will be duly observed. Therefore, it is essential that the concentrations of siRNAs are measured correctly. When doing so, one must consider not only what the apparent concentrations are by OD260 reading, but also whether the RNA strands are of full-length and whether only dsRNA molecules are counted. This issue might not affect data interpretation if appropriate controls are included in one set of RNAi experiments, but it could have significant influence on conclusions if data from different experiment sets or labs are compared or combined.
HPLC purification currently provides the best means to remove RNA molecules with deletions or remain single-stranded, however, the price tag added by most reagent providers for such treatment has been prohibiting because manufacturers either need to start synthesis at a much bigger scale to obtain promised amount, or they do not promise the delivery quantity at all. The phosphoramidites (oligo building blocks) for RNA synthesis can be 10 times or more expensive than for DNA. Some companies offer alternative purification methods such as a cartridge type device, but they can only remove salt and small impurities, not RNA oligos of shorter lengths accumulated at each cycle of amide coupling. The AllHPLC siRNAs within Allele’s RNAi product line, pre-validated or custom made, are uniformly HPLC purified with 5 OD or 12.5 nmol of double-stranded, annealed siRNA delivered. Allele passes to customers the cost savings from manufacturing our own RNA amidites and other reagents for oligo synthesis. The pre-validated HPLC purified double-stranded siRNA is offered today at $149/12.5 nmol.
Before purchasing siRNAs, even at a low cost of $29 per pair of HPLC purified control siRNA from Allele, researchers still need to consider how well their cells can be transfected. For hard-to-transfect cells, lentiviral vectors carrying a shRNA expressing cassette is often a better choice. To establish stable cell lines, plasmid vectors should be considered. For low cost target screening, the PCR format linear siRNA expression cassettes have advantages.
Tuesday, September 15, 2009
Publication by Allele Scientists: A Tale of Twin Functions
A paper published by PLoS One on 08-15-09 demonstrated one RNA binding factor can bind to two completely different target RNA sequences while play leading roles in general splicing machinery and backup roles in alternative pre-mRNA splicing.
In Drosophila, sans-fille (snf) encodes a ~25-kDa protein that was found to carry the functions of two human proteins U1A and U2B”, components of general splicing machinery U1 and U2 snRNPs (small nuclear ribonucleoproteins), respectively. The SNF protein is able to do the double duties by binding to U1 RNA, the RNA component of the U1snRNP complex alone, and the U2 RNA of U2 snRNP if helped by another U2 snRNP -specific protein.
Busy as it sounds, snf has also been known for a long time to be a major participant of the fly sex determination pathway. When the master sex determination gene Sex-lethal (Sxl) is weakened by mutation or in the germ line before SXL fully takes over sex-specific development, snf is required to maintain appropriate splicing patterns of sex-specific transcripts such as Sxl.
How does a general splicing machinery component work specifically in regulated tissue-specific alternative splicing events?
The PLoSOne paper by researchers from Allele Biotech in San Diego and Peking University in China showed that the SNF protein can directly bind to elements of the Sxl pre-mRNA, in a way different from its binging to U1 RNA. Like many RRM (RNA Recognition Motif)- or RBD (RNA Binding Domain)-containing RNA binding proteins, SNF has more than one such conserved RNA binding motifs. Hu et al. in Bin Xia’s lab showed that SNF uses just one RRM to bind to U1 snRNA, but both RRMs to bind to consecutive U-bases on the Sxl pre-mRNA. The result was obtained through NMR analysis of SNF bound to RNAs. This structure-based conclusion agrees well with molecular biology results that showed a longer than usual RNA segment, e.g. ~16 U-bases, is needed for SNF to bind by Jiwu Wang, a co-senior author of the publication.
The long U-runs can be separated into two 8 U-base segments, each perhaps recognized by one RRM. More interestingly, from a mechanical point of view, the two segments can be separated by at least 120-nucleotide distance and still function as effective SNF binding substrate. Functionally, given that many ~8 U-runs surround the alternative splice site on the Sxl pre-mRNA, which are shown by Jiwu Wang’s earlier publications, to be cooperative binding site for the master sex determination factor SXL, it is intriguing to model that SNF binds to these same RNA elements for the same functions. But instead of binding to 2 U-runs cooperatively (therefore much more strongly) as SXL does, it needs 2 runs to just bind to the alternative splice sites. But when SNF does bind, the two U-runs may be used to loop out an entire region of RNA (such as an exon) by one SNF protein, providing effective regulatory functions. This could give a nearly perfect explanation as how SNF can help sex determination only when needed.
For more similar news: http://www.allelebiotech.com/News, or blogs http://allelebiotech.com/blogs/
Hu et al.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0006890#aff4
In Drosophila, sans-fille (snf) encodes a ~25-kDa protein that was found to carry the functions of two human proteins U1A and U2B”, components of general splicing machinery U1 and U2 snRNPs (small nuclear ribonucleoproteins), respectively. The SNF protein is able to do the double duties by binding to U1 RNA, the RNA component of the U1snRNP complex alone, and the U2 RNA of U2 snRNP if helped by another U2 snRNP -specific protein.
Busy as it sounds, snf has also been known for a long time to be a major participant of the fly sex determination pathway. When the master sex determination gene Sex-lethal (Sxl) is weakened by mutation or in the germ line before SXL fully takes over sex-specific development, snf is required to maintain appropriate splicing patterns of sex-specific transcripts such as Sxl.
How does a general splicing machinery component work specifically in regulated tissue-specific alternative splicing events?
The PLoSOne paper by researchers from Allele Biotech in San Diego and Peking University in China showed that the SNF protein can directly bind to elements of the Sxl pre-mRNA, in a way different from its binging to U1 RNA. Like many RRM (RNA Recognition Motif)- or RBD (RNA Binding Domain)-containing RNA binding proteins, SNF has more than one such conserved RNA binding motifs. Hu et al. in Bin Xia’s lab showed that SNF uses just one RRM to bind to U1 snRNA, but both RRMs to bind to consecutive U-bases on the Sxl pre-mRNA. The result was obtained through NMR analysis of SNF bound to RNAs. This structure-based conclusion agrees well with molecular biology results that showed a longer than usual RNA segment, e.g. ~16 U-bases, is needed for SNF to bind by Jiwu Wang, a co-senior author of the publication.
The long U-runs can be separated into two 8 U-base segments, each perhaps recognized by one RRM. More interestingly, from a mechanical point of view, the two segments can be separated by at least 120-nucleotide distance and still function as effective SNF binding substrate. Functionally, given that many ~8 U-runs surround the alternative splice site on the Sxl pre-mRNA, which are shown by Jiwu Wang’s earlier publications, to be cooperative binding site for the master sex determination factor SXL, it is intriguing to model that SNF binds to these same RNA elements for the same functions. But instead of binding to 2 U-runs cooperatively (therefore much more strongly) as SXL does, it needs 2 runs to just bind to the alternative splice sites. But when SNF does bind, the two U-runs may be used to loop out an entire region of RNA (such as an exon) by one SNF protein, providing effective regulatory functions. This could give a nearly perfect explanation as how SNF can help sex determination only when needed.
For more similar news: http://www.allelebiotech.com/News, or blogs http://allelebiotech.com/blogs/
Hu et al.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0006890#aff4
Wednesday, September 9, 2009
iPS Cells: Feeder Cells
Allele’s entire iPSC product line is designed for the ease of the researcher. Each component in our iPSC catalog will shave priceless time off your protocol by eliminating the tedious steps in iPS induction so you can get down to work.
Allele is adding a major component to its iPSC line: pre-irradiated, ready-to-use, system specific, bFGF-Producing Feeder Cells for iPSC propagation!
Using Allele’s bFGF-Producing Feeder Cells avoids the usual problems associated with MEF cell lines. They are maintained at low passages, come pre-irradiated and ectopically express bFGF so there is no need to supplement your medium with additional growth factors.
Additionally, Allele Biotech is introducing human fibroblasts to the market for iPSC work. MEF is good for mouse iPSC reprogramming but human fibroblast feeders are preferred when creating human iPSCs due to their secreted factors. Propagate human iPSC with greater efficiency while eliminating non-human cells for therapeutic use of human iPSCs!
As always we encourage customer feed back. We are interested to hear about your stem cell work, needs, and requests for new products. We also welcome those who have new ideas and potential products to collaborate with us. We are here to help advance your research and get your technologies to the public.
If you are enjoying AlleleNews and AlleleBlogs: come back and check out our new Forum and FAQ Sections soon to be added to our blogs for quick product/service related exchange and messages of more user control.
Allele is adding a major component to its iPSC line: pre-irradiated, ready-to-use, system specific, bFGF-Producing Feeder Cells for iPSC propagation!
Using Allele’s bFGF-Producing Feeder Cells avoids the usual problems associated with MEF cell lines. They are maintained at low passages, come pre-irradiated and ectopically express bFGF so there is no need to supplement your medium with additional growth factors.
Additionally, Allele Biotech is introducing human fibroblasts to the market for iPSC work. MEF is good for mouse iPSC reprogramming but human fibroblast feeders are preferred when creating human iPSCs due to their secreted factors. Propagate human iPSC with greater efficiency while eliminating non-human cells for therapeutic use of human iPSCs!
As always we encourage customer feed back. We are interested to hear about your stem cell work, needs, and requests for new products. We also welcome those who have new ideas and potential products to collaborate with us. We are here to help advance your research and get your technologies to the public.
If you are enjoying AlleleNews and AlleleBlogs: come back and check out our new Forum and FAQ Sections soon to be added to our blogs for quick product/service related exchange and messages of more user control.
Wednesday, September 2, 2009
Allele Annouces New Products Based on Camelid Antibodies
090209 San Diego—Allele Biotech formally announced today that it has signed an agreement to distribute products from ChromoTek GmbH, a German company with a focus on camelid antibody fragment based precipitation and detection reagents. Single-domain antigen binding fragment, also called VHH or nanobody, can be derived from heavy chain-only antibodies produced by animals in the camel family. The small size and special structures of VHH enable their efficient binding into areas not normally accessible to larger IgG antibodies. Although GFP is a commonly used tag in fusion proteins for imaging, it has not yet become a widely used tag for precipitation. With the introduction of the first VHH-based research reagent GFP-Trap, GFP-fusions will become a desirable tool for pulldown. GFP-Trap is immobilized anti-GFP nanobody, which with a simple procedure, can result in quantitative depletion or isolation of GFP-fusions.
Applications of GFP-Trap may include ChIP-CHIP, CLIP, co-IP, enzyme activity analysis (see Allele Biotech’s product group main page for sample publications). Other products such as anti-RFP and anti-GFP monoclonal antibodies that may be used after GFP-fusion precipitation are now also available from Allele Biotech. “VHH fragments have great potentials in both therapeutic and basic research,” said Allele’s CEO Dr. Jiwu Wang, “The agreement will significantly strengthen Allele Biotech's position in the antibody field”. Allele Biotech started with a grant from the NIH in 2000 to develop ways to display and select antibodies. It participated in a collaborative project on yeast display for selecting antibodies against cancer antigens in 2007 for the NCI. After acquiring Orbigen in 2008, Allele has thousands of antibodies in its product line.
Applications of GFP-Trap may include ChIP-CHIP, CLIP, co-IP, enzyme activity analysis (see Allele Biotech’s product group main page for sample publications). Other products such as anti-RFP and anti-GFP monoclonal antibodies that may be used after GFP-fusion precipitation are now also available from Allele Biotech. “VHH fragments have great potentials in both therapeutic and basic research,” said Allele’s CEO Dr. Jiwu Wang, “The agreement will significantly strengthen Allele Biotech's position in the antibody field”. Allele Biotech started with a grant from the NIH in 2000 to develop ways to display and select antibodies. It participated in a collaborative project on yeast display for selecting antibodies against cancer antigens in 2007 for the NCI. After acquiring Orbigen in 2008, Allele has thousands of antibodies in its product line.
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