Müller was awarded the Nobel prize for her depictions of the "landscape of the dispossessed" in novels that demonstrate "the concentration of poetry and the frankness of prose". Born in Romania, the author received death threats for refusing to become an informant for President Nicolae Ceausescu's department of state security, the Securitate, and eventually emigrated to Germany.
Original report by Alison Flood and published Guardian.UK
Müller's lecture on winning the Nobel saw her lay out in detail the moment when the Securitate attempted to force her to sign a document describing herself as a collaborator. "He called me stupid, said I was a shirker and a slut, as corrupted as a stray bitch," Müller said. "Without sitting down, I wrote what he dictated my name, date of birth and address. Next, that I would tell no one, no matter how close a friend or relative, that I ... and then came the terrible word: colaborez I am collaborating. At that point I stopped writing."
She told the man that she didn't "have the character for this". "The word character made the Securitate man hysterical," she said. "He tore up the sheet of paper and threw the pieces on the floor. Then he probably realized he would have to show his boss that he had tried to recruit me, because he bent over, picked up the scraps and tossed them into his briefcase. After that he gave a deep sigh and, defeated, hurled the vase with the tulips against the wall. As it shattered it made a grinding sound, as though the air had teeth. With his briefcase under his arm he said quietly: 'You'll be sorry, we'll drown you in the river'. I said as if to myself: 'If I sign that, I won't be able to live with myself anymore, and I'll have to do it on my own. So it's better if you do it'."
Monday, March 22, 2010
Friday, March 19, 2010
Fluorescent Protein-Based Assay Development
This blog will be the template of Allele's new cell based assay service landing page. http://www.allelebiotech.com/allele3/index.php
Overview:
Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.
Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.
FPs as fusion tags:
Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.
FPs as expression reporters:
FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.
Overview:
Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.
Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.
FPs as fusion tags:
Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.
FPs as expression reporters:
FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.
New Product of the Week 03-15-10 to 03-21-10: Oct4-Sox2 2-in-1 lentivirus ABP-SC-LVI2in1 for effective iPS generation link: http://www.allelebiotech.com/shopcart/index.php?c=132&sc=122.
Promotion of the Week 03-15-10 to 03-21-10: 5% off plate oligos at all scales! www.allelebiotech.com/allele3/Oligo_96Plate.php We are doing our “window promotion” again, during a hour-long window, get any Allele’s High efficiency competent cells at 30% regular price, the time will be announced tomorrow on our Facebook page.
Overview:
Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.
Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.
FPs as fusion tags:
Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.
FPs as expression reporters:
FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.
Overview:
Originally cloned from the jellyfish Aequorea victoria and subsequently from many other marine organisms, fluorescent proteins (FPs) spanning the entire visual spectrum have become some of the most widely used genetically encoded tags. Unlike traditional labeling methods, FPs may be used to specifically label virtually any protein of interest in a living cell with minimal perturbation to its endogenous function. Genes encoding FPs alone or as fusions to a protein of interest may be introduced to cells by a number of different methods, including simple plasmid transfection or viral transduction. Once expressed, FPs are easily detected with standard fluorescence microscopy equipment.
Factors that should be taken into account when designing an FP-based imaging experiment include the desired wavelength(s) for detection, the pH environment of the tagged protein, the total required imaging time, and the expression level or dynamic range required for detection of promoter activity or tagged protein. Individual FPs currently available to the research community vary considerably in their photostability, pH sensitivity, and overall brightness, and so FPs must be chosen with care to maximize the likelihood of success in a particular experimental context.
FPs as fusion tags:
Use of FPs as fusion tags allows visualization of the dynamic localization of the tagged protein in living cells. For such applications, the cDNA of a protein of interest is attached in-frame to the coding sequence for the desired FP, and both are put under the control of a promoter appropriate to the experimental context (typically CMV for high-level expression, though other promoters may be desirable if overexpression of your protein of interest is suspected of producing artifacts). The most basic uses for fluorescent protein fusions include tracking of specific organelles (fusions to short organelle targeting signals) or cytoskeletal structures (fusions to actin or tubulin, for example). More advanced uses include tracking receptors or exported proteins. In most cases, it is critical that the FP used for fusion tagging be fully monomeric, as any interaction between fusion tags is likely to produce artifacts, some of which may be hard to recognize in the absence of other controls. While in most cases FP fusions do not interfere with normal protein function, whenever possible, FP fusion proteins should be validated by immunostaining the corresponding endogenous protein in non-transfected cells and verifying similar patterns of localization.
FPs as expression reporters:
FPs are highly useful as quantitative expression reporters. By driving the expression of an FP gene by a specific promoter of interest, it is possible to produce an optical readout of promoter activity. Use of the brightest possible FP ensures the best dynamic range for such an experiment. Because dynamic localization is not generally an issue for expression reporter applications, it is possible to use non-monomeric FPs for this purpose, opening up additional possibilities for multiple wavelength imaging. In order to obtain more reliable quantitative data and to correct for likely variations between individual cells in expression reporter experiments, the use of two spectrally distinct (e.g. green and red) FPs is advisable. By driving expression of one FP with a constitutive promoter and a second FP with the promoter of interest, the ratio of the two signals provides a quantitative readout of relative activity. Averaged over many cells, this technique should provide statistical power necessary for quality expression level experiments. Because FPs normally have a very slow turnover rate in mammalian cells, it may be desirable to add a degradation tag to your FP to enhance temporal resolution when measuring highly dynamic promoter activity.
New Product of the Week 03-15-10 to 03-21-10: Oct4-Sox2 2-in-1 lentivirus ABP-SC-LVI2in1 for effective iPS generation link: http://www.allelebiotech.com/shopcart/index.php?c=132&sc=122.
Promotion of the Week 03-15-10 to 03-21-10: 5% off plate oligos at all scales! www.allelebiotech.com/allele3/Oligo_96Plate.php We are doing our “window promotion” again, during a hour-long window, get any Allele’s High efficiency competent cells at 30% regular price, the time will be announced tomorrow on our Facebook page.
Thursday, March 11, 2010
How I started my company and why-Jiwu Wang's speech at SDEE's naugural event
Originally presented at the first ever SDEE meeting held at Sanford-Burhnam Institute.
I started Allele Biotech when I was a fourth year postdoc at UCSD, just finished my postdoc fellowship. I wish I could say it was all planned out with clear goals and measurable milestones, as how I would like to describe everything we do these days, but it was not. Actually I had all the reasons not to start a company. My background, training and interactions with my mentors and colleagues really set my mind on an academic career path, and I had a pretty good publication record and a hint of an invitation from a former committee member to apply for a faculty job. On the other hand, I had no experience at all or much interest in business.
The one factor that did push me in the direction of becoming an entrepreneur was that I was exposed to the fact that small business could get NIH grants through an application, review and award process not too different from that of a typical research grant. That is something I kind knew how to do and had interest to try. I had friends who were truly interested in business and wanted to get their feet wet in a startup before moving on to higher positions in bigger places. We had no way of attracting real investment though. If I were an angel or venture investor I wouldn’t invest in ourselves since we were all postdocs with no experience running any serious business. So I started doing the one thing I did learn how to do through years in academic labs, writing grants, and my publication record didn't hurt. The reviewers said this is a first rate scientist with a good idea, let’s give him a chance.
The core of these grants were basically something I knew very well, such as RNA molecules, polymerase, protein-RNA interactions, and they were presented in such a way that suggested the research results could be turned into something useful for advancing biology. Well, I got my first grant, then the second, and the third…that was how we had the company up and running. To be a PI on a business research grant you need to be above 50% employed by the company, so even though I wasn’t committed to the company and didn’t draw any salary from it for a long while, I had to request a demotion to 49% status at UCSD and lived on a half postdoc pay.
The above is how I started my company, here comes why I am still there. The number one reason that I gradually moved to be completely dedicated to the company, believe it or not, was academic freedom, by which I mean you can run wild with any ideas for an experiment and don’t have to worry about any pressure from anybody, as long as you take responsibility for all the consequences. To me, research in a small business setting provides the ultimate freedom in the broadest sense. We got our first product two years after the company started, a patent filing on RNA interference which was outlicensed to Promega for marketing.
A couple more grants and patent filings later we have built a critical mass in terms of products, company structure, and a gradual understanding of the difference between pure research and running a business. In 04 we started direct marketing of research reagents in siRNA and genotyping, in 08 we acquired a company that was bigger and older than us, and added more than 1,000 products in the fields of antibodies and viral systems. We were hit pretty hard by the current economic downturn, and we pulled ourselves out by the hair focusing on two things: online presence and adding new products. Even in the summer of 09 when we were in the deepest financial trouble, we did not make any dramatic changes in our employment situations, but rather, we added one new product line per month in the field of stem cell/iPS, fluorescent proteins, biosensors, screening services, etc, at least one new product per week. We are very active in social networking, having daily activities in Facebook, Myspace, Twitter, Linkedin, etc. That’s where we are now.
Biotech is a fast moving field, we should never run out of ideas, rather our challenge is to build products around new discoveries and find a balance between trailblazing and completing what we have started.
I started Allele Biotech when I was a fourth year postdoc at UCSD, just finished my postdoc fellowship. I wish I could say it was all planned out with clear goals and measurable milestones, as how I would like to describe everything we do these days, but it was not. Actually I had all the reasons not to start a company. My background, training and interactions with my mentors and colleagues really set my mind on an academic career path, and I had a pretty good publication record and a hint of an invitation from a former committee member to apply for a faculty job. On the other hand, I had no experience at all or much interest in business.
The one factor that did push me in the direction of becoming an entrepreneur was that I was exposed to the fact that small business could get NIH grants through an application, review and award process not too different from that of a typical research grant. That is something I kind knew how to do and had interest to try. I had friends who were truly interested in business and wanted to get their feet wet in a startup before moving on to higher positions in bigger places. We had no way of attracting real investment though. If I were an angel or venture investor I wouldn’t invest in ourselves since we were all postdocs with no experience running any serious business. So I started doing the one thing I did learn how to do through years in academic labs, writing grants, and my publication record didn't hurt. The reviewers said this is a first rate scientist with a good idea, let’s give him a chance.
The core of these grants were basically something I knew very well, such as RNA molecules, polymerase, protein-RNA interactions, and they were presented in such a way that suggested the research results could be turned into something useful for advancing biology. Well, I got my first grant, then the second, and the third…that was how we had the company up and running. To be a PI on a business research grant you need to be above 50% employed by the company, so even though I wasn’t committed to the company and didn’t draw any salary from it for a long while, I had to request a demotion to 49% status at UCSD and lived on a half postdoc pay.
The above is how I started my company, here comes why I am still there. The number one reason that I gradually moved to be completely dedicated to the company, believe it or not, was academic freedom, by which I mean you can run wild with any ideas for an experiment and don’t have to worry about any pressure from anybody, as long as you take responsibility for all the consequences. To me, research in a small business setting provides the ultimate freedom in the broadest sense. We got our first product two years after the company started, a patent filing on RNA interference which was outlicensed to Promega for marketing.
A couple more grants and patent filings later we have built a critical mass in terms of products, company structure, and a gradual understanding of the difference between pure research and running a business. In 04 we started direct marketing of research reagents in siRNA and genotyping, in 08 we acquired a company that was bigger and older than us, and added more than 1,000 products in the fields of antibodies and viral systems. We were hit pretty hard by the current economic downturn, and we pulled ourselves out by the hair focusing on two things: online presence and adding new products. Even in the summer of 09 when we were in the deepest financial trouble, we did not make any dramatic changes in our employment situations, but rather, we added one new product line per month in the field of stem cell/iPS, fluorescent proteins, biosensors, screening services, etc, at least one new product per week. We are very active in social networking, having daily activities in Facebook, Myspace, Twitter, Linkedin, etc. That’s where we are now.
Biotech is a fast moving field, we should never run out of ideas, rather our challenge is to build products around new discoveries and find a balance between trailblazing and completing what we have started.
Sunday, March 7, 2010
近日疯传的段子, 一笑
Weekend laughs, even though maybe not so funny for those who live in the jokes
1 、检讨书
我刚到纪委工作就犯了严重错误：前天我接到通知说中央电视台记者要来采访我市廉政先进典型，领导要我通知几个好局长来。
为了赶快下班，所以我通知各单位时说的比较简单：“请你们局长明天到纪委来一趟。”
没想到国土局长接到通知后大小便失禁，心脏病突发，不醒人事。财政局长自首了。交通局长当晚就失踪，据说已逃往加拿大。工商局长连夜杀死情妇，他以为她出卖了他。卫生局长服毒自杀，还留下检举别人的名单。
这惨痛的一切都是我工作方法简单造成的，我痛定思痛，深感内疚，特作检讨！
2 、密码
深圳市市长许宗衡被双规，家中一大保险柜久不能开。一识货中纪委官员说：此乃声控锁，密码多用八个字。
于是办案人员召集大家轮流猜试 : “人不为己，天诛地灭”; “芝麻开门，芝麻开门”; “上天保佑，升官发财”; “八仙过海，各显神通”; “人为财死，鸟为食亡”; ……但均不灵!
无奈，押许市长至此。他清清嗓子，用浓重的湖南湘潭话正色道：“清正廉洁，执政为民！”柜门应声而开，满柜金银珠宝惊呆众人!
3. 书记
过去中国出国的考察团都让书记当团长。翻译见外国人后介绍此人是总工、此人是技术员……此人是考察团团长--X书记。外国人不知道什么叫书记，翻译说就是做思想工作的人。外国人说原来是类似神父牧师之类的人。翻译说不对，它的思想工作是告诉大家社会主义一定胜利，资本主义一定灭亡的。外国人听了恍然大悟，说明白了，原来是骗子。
1 、检讨书
我刚到纪委工作就犯了严重错误：前天我接到通知说中央电视台记者要来采访我市廉政先进典型，领导要我通知几个好局长来。
为了赶快下班，所以我通知各单位时说的比较简单：“请你们局长明天到纪委来一趟。”
没想到国土局长接到通知后大小便失禁，心脏病突发，不醒人事。财政局长自首了。交通局长当晚就失踪，据说已逃往加拿大。工商局长连夜杀死情妇，他以为她出卖了他。卫生局长服毒自杀，还留下检举别人的名单。
这惨痛的一切都是我工作方法简单造成的，我痛定思痛，深感内疚，特作检讨！
2 、密码
深圳市市长许宗衡被双规，家中一大保险柜久不能开。一识货中纪委官员说：此乃声控锁，密码多用八个字。
于是办案人员召集大家轮流猜试 : “人不为己，天诛地灭”; “芝麻开门，芝麻开门”; “上天保佑，升官发财”; “八仙过海，各显神通”; “人为财死，鸟为食亡”; ……但均不灵!
无奈，押许市长至此。他清清嗓子，用浓重的湖南湘潭话正色道：“清正廉洁，执政为民！”柜门应声而开，满柜金银珠宝惊呆众人!
3. 书记
过去中国出国的考察团都让书记当团长。翻译见外国人后介绍此人是总工、此人是技术员……此人是考察团团长--X书记。外国人不知道什么叫书记，翻译说就是做思想工作的人。外国人说原来是类似神父牧师之类的人。翻译说不对，它的思想工作是告诉大家社会主义一定胜利，资本主义一定灭亡的。外国人听了恍然大悟，说明白了，原来是骗子。
Friday, March 5, 2010
How to Generate Conditional Knockout Mice with Cre
From AlleleBlog: http://allelebiotech.com/blogs/2010/03/how-to-generate-conditional-knockout-mice-with-cre/
The bacterial Cre recombinase targets a specific DNA sequence called loxP and deletes a segment of DNA flanked by loxP sequences. This system is often used in the generation of knockout and conditional knockout animals.
The knockout of specific genes leading to embryonic lethal phenotype will not yield adult animals. Cre-lox recombination provides a means to knockout the specific genes in adult mice, or to introduce a knockout phenotype in specific tissues (conditional knockout) using tissue-specific promoter driven Cre or an inducible Cre.
The cutting by Cre at the loxP sites and rejoining by ligase is an efficient process. During this process, inverted loxP sites will result in an inversion, whereas direct repeat will cause a deletion. Cre/lox recombination is a one-way reaction so there is no need for continued Cre expression. Therefore, Cre can be introduced by adenovirus or lenti/retrovirus. Here is an example of using adnovirus-Cre in one lab: for MEF, on a 70% confluent P10 cm plate (probably 2-2.5 million cells), use 6ul of 1.1×10^12 adenovirus-Cre, which will give 80% infection; or use 10ul of 1.1×10^12 adenovirus-Cre to get 90% infection, with GFP as marker and analyzed by FACS.
Adenovirus could post a toxicity problem when used at the very high titers to reach high percentage of transduction. An alternative is to use only lentivirus-Cre, at only about 1-2 ul and still obtain >80% infection. However, a silencing event needs to occur before the expression of Cre from lentivirus is shut off. The timing and degree of silencing is not controlled in such experiments. Continued expression of Cre should not influence most experiments.
To be certain that the Cre enzyme can be successfully delivered into the nucleus for conditional knockout to occur, the bacterial Cre gene needs to be engineered to contain a nuclear localization (nl) signal of eukaryotic cells. The function of the nuclear-localized Cre (nlCre) can be tested using a loxP-nuclear localized lacZ (nlacZ) reporter cell line, which can be used to monitor the function of the nlCre recombinase.
New Product of the Week 03-01-10 to 03-07-10: Lentivirus-Cre ABP-RP-TLCCreS and ABP-RP-TLCCreL, link: http://www.allelebiotech.com/shopcart/index.php?c=219&sc=188.
Promotion of the Week 03-01-10 to 03-07-10: for a limited time, get lab agarose at ~1/5 of the big company’s price. http://www.facebook.com/#!/pages/San-Diego-CA/Allele-Biotechnology-and-Pharmaceuticals-Inc/78331924957
The bacterial Cre recombinase targets a specific DNA sequence called loxP and deletes a segment of DNA flanked by loxP sequences. This system is often used in the generation of knockout and conditional knockout animals.
The knockout of specific genes leading to embryonic lethal phenotype will not yield adult animals. Cre-lox recombination provides a means to knockout the specific genes in adult mice, or to introduce a knockout phenotype in specific tissues (conditional knockout) using tissue-specific promoter driven Cre or an inducible Cre.
The cutting by Cre at the loxP sites and rejoining by ligase is an efficient process. During this process, inverted loxP sites will result in an inversion, whereas direct repeat will cause a deletion. Cre/lox recombination is a one-way reaction so there is no need for continued Cre expression. Therefore, Cre can be introduced by adenovirus or lenti/retrovirus. Here is an example of using adnovirus-Cre in one lab: for MEF, on a 70% confluent P10 cm plate (probably 2-2.5 million cells), use 6ul of 1.1×10^12 adenovirus-Cre, which will give 80% infection; or use 10ul of 1.1×10^12 adenovirus-Cre to get 90% infection, with GFP as marker and analyzed by FACS.
Adenovirus could post a toxicity problem when used at the very high titers to reach high percentage of transduction. An alternative is to use only lentivirus-Cre, at only about 1-2 ul and still obtain >80% infection. However, a silencing event needs to occur before the expression of Cre from lentivirus is shut off. The timing and degree of silencing is not controlled in such experiments. Continued expression of Cre should not influence most experiments.
To be certain that the Cre enzyme can be successfully delivered into the nucleus for conditional knockout to occur, the bacterial Cre gene needs to be engineered to contain a nuclear localization (nl) signal of eukaryotic cells. The function of the nuclear-localized Cre (nlCre) can be tested using a loxP-nuclear localized lacZ (nlacZ) reporter cell line, which can be used to monitor the function of the nlCre recombinase.
New Product of the Week 03-01-10 to 03-07-10: Lentivirus-Cre ABP-RP-TLCCreS and ABP-RP-TLCCreL, link: http://www.allelebiotech.com/shopcart/index.php?c=219&sc=188.
Promotion of the Week 03-01-10 to 03-07-10: for a limited time, get lab agarose at ~1/5 of the big company’s price. http://www.facebook.com/#!/pages/San-Diego-CA/Allele-Biotechnology-and-Pharmaceuticals-Inc/78331924957
Wednesday, March 3, 2010
How big is an 8.8 earthquake? After reading a Chinese article on US news media response
Yes 8.8 earthquake means 10 times more violent than 7.8, but no it does mean "太子港和汶川毁灭十次了吗". The degree of damage is related but not the same as the H.F. Reid's measurement. ~700-800 people died in Chile, a terrible tragedy but still 100-200 times less than Wenchuan 汶川 in China 2 years ago, and 300-400 times less than Haiti months ago.
I don't see what is wrong with bystanders in 夏威夷 "显然也都像笔者同样急切地想见证一个庄严历史时刻的到来", I took my kids to a high point near home overlooking San Diego beach to catch the possible wave, I didn't feel it 庄严 or anything, just a moment seldom occurs. The author seems to use this irony to underline the main point "甚至对即将发生在夏威夷的海啸，也因为远离美国本土，而不大会引起美国媒体太大的兴趣，从而影响他们享受周末烧烤的情绪". Just because you want to watch it 24/7, does not mean others should feel the same or the medium suits your needs. Yes, I did my weekend BBQ too, what the heck is wrong with that? Did you have your lunch and dinner, what was it? Noodles? Is that less disgusting in such an "historical event?" than BBQ, why didn't you skip all the meals since everything in Chile could have been completely leveled?
Talking about the US response, the president went on TV to give condolences and promise to help (US promise to help has always been carried out) the same day, LA urban search and rescue team of hundreds of professionals were on flight readiness a day after, and only a few days back from Haiti, with the knowledge that LA is a high danger area that might need them at any moment too and that's why their team was built with US and CA tax dollars. Secretary Clinton changed trip plan to stop by Chile days later and brought with her material help. Have to disagree with the following "相比一个月前的海地地震，这次更为强烈的地震没有引起美国媒体的迅速关注，显然也有“灾难疲劳”的原因。自然灾难如果频繁发生，人们对其就不再敏感，甚至会无动于衷，正如在战争中人们适应了频繁的轰炸杀戮之后，只要尚未危及自身也会不再害怕，对苦难变得迟钝甚至无动于衷一样。
“事不关己，高高挂起，”笔者心中突然冒出这样一句话来，不知是悲哀，还是嘲讽。不是这样吗？正当圣地亚哥一片火海，遍地狼藉，全国各地上百万所建筑成为无法居住的危房时，夏威夷人却涌向海边，架上摄像机，一副见证盛典或奇迹，抑或一睹也许是百年不遇的巨浪风采。" I don't know how you 事不关己, 低低坐地， I live my life to the best I can, every god-dxxm day, including that eventful day as you see it, and I and my whole family including the history watching kids of 5 and 6, donated money.
This 兴无灭资 has a real clogged view by the ridiculous 无 thinking, because the pen name 笔者心中突然冒出这样一句话来 it sounded so much like 0.5 thinking though.
______________________________
Original author ·兴无灭资·
今天是星期六，醒来第一件事自然是打开床边椅子上的手提电脑，查一下电邮。一打开电邮首页，一副触目惊心的照片跃入眼帘。只见一座坍塌的立交桥下，底朝天地躺着四五辆轿车和载货卡车，就像一具具肢体残缺的尸体，惨不忍睹。再一看标题，才知道自己还在梦乡的时候，智利刚刚发生了一场8.8级的地震。
8.8级，这是一个什么概念？据说在地震仪上，每上升一级，地震强度就增加十倍。最近海地的地震，包括前年的汶川地震，都不超过8度。所以8.8度就意味着十倍的海地和汶川地震，那不早就把太子港和汶川毁灭十次了吗？
但是这个想法很快就被接下来阅读电邮的过程打断，甚至很快就抛掷脑后，不再多想了。直到下楼吃午饭时，打开电视用遥控选台时，才发现一家西班牙语台显然已经对此连续报道多时了。只见主播一会儿连线现场的记者，一会儿连线墨西哥城的一家电视台的演播室，不断在互通情报。同时，电视画面不停地变换着视频和照片，展示智利首都圣地亚哥的受损情况。由于地震发生在海边，所以会引起了太平洋沿岸的海啸。因此，半个小时后，镜头转向了夏威夷。主播甚至连线到当地的居民，向他们询问哪里是否发生了什么状况，并把镜头锁定在夏威夷旅游胜地的一片海滩上。同时，电视屏幕的下方出现了两行文字，说数小时内（solas horas）海啸就会到达夏威夷。笔者离开了电机一会儿，回来就发现文字已经变成了数分钟内（solos minutos）海啸就会到达夏威夷了。这把笔者吓得赶快坐回到沙发上，急切地等待着这一历史时刻的到来。
只是左等右等，看到的只是沙滩岸边的高地和堤坝上聚集的人群越来越大，显然也都像笔者同样急切地想见证一个庄严历史时刻的到来。但是，目光所至，并不见海啸到来的迹象，有的只是几个不畏警告，仍在风口浪尖上弄潮的冲浪者和点缀在蓝白波涛之上的白色风帆。
显然，电视台为了不失去观众，不惜玩弄文字游戏以达到操纵观众的目的。但是笔者对这家电视台放弃既定节目和所有广告，持续跟踪报道正在发生的新闻事件，以满足有关观众知情权的态度，不得不表示欣赏。在等待的过程中，笔者浏览了一下其它电视台的节目，发现除了德国之声正在自己的新闻节目中对此进行一般报道之外，没有一家美国英文电台在报道这个突发新闻。它们的周末电影依旧照常进行。显然，发生在几千英里之外一个南美国家的突发灾难并没没有足够的理由中断美国人惬意的周末。甚至对即将发生在夏威夷的海啸，也因为远离美国本土，而不大会引起美国媒体太大的兴趣，从而影响他们享受周末烧烤的情绪。
结果，笔者只好守住这家西班牙语台，权当练习听力了。由此可见， “血浓于水”的道理，普世皆适。移民美国的智利人和其他南美人，肯定非常关心自己仍在故国的亲人，一定都把眼睛粘在这个频道上了。同时，通过这家电视台呼吁的“帮助智利”捐款活动，已经展开。
相比一个月前的海地地震，这次更为强烈的地震没有引起美国媒体的迅速关注，显然也有“灾难疲劳”的原因。自然灾难如果频繁发生，人们对其就不再敏感，甚至会无动于衷，正如在战争中人们适应了频繁的轰炸杀戮之后，只要尚未危及自身也会不再害怕，对苦难变得迟钝甚至无动于衷一样。
“事不关己，高高挂起，”笔者心中突然冒出这样一句话来，不知是悲哀，还是嘲讽。不是这样吗？正当圣地亚哥一片火海，遍地狼藉，全国各地上百万所建筑成为无法居住的危房时，夏威夷人却涌向海边，架上摄像机，一副见证盛典或奇迹，抑或一睹也许是百年不遇的巨浪风采。
不知什么原因，笔者脑中浮现出《动物世界》中经常出现的镜头：一只老虎或者狮子，一头冲进大群野牛或野鹿丛中，单枪匹马地扑倒其中的一员，然后从容地把它拖到草丛中去从容地享用。而受害者的同类，哪怕成千上万，却只顾东奔西窜，各自落荒而逃。笔者经常天真地假设，它们也无需有意识地团结起来，那样做要求也过高了一些；它们只需依着本能，掉过头来冲着对手做一个简单的stampede，就足以将其碾成灰烬。
同理，人类在面对天灾人祸时，也不必一定要有意识地撇开意识形态的分野和政经利益的博弈，而只需凭着生存的本能，集体地面对危险的挑战。尤其是天灾的危险，对任何人都是一视同仁的，不会因为你是腰缠万贯的富翁就放你一马。于是，为了避免让天灾人祸将人类各个击破，人类哪怕只是为了一己的安危，也应放弃前嫌，忘记各自的种族，宗教，经济和政治的区别和利益，共同应对诸如气候变化，资源匮乏，和环境污染等会致整个人类于死地的难题。
I don't see what is wrong with bystanders in 夏威夷 "显然也都像笔者同样急切地想见证一个庄严历史时刻的到来", I took my kids to a high point near home overlooking San Diego beach to catch the possible wave, I didn't feel it 庄严 or anything, just a moment seldom occurs. The author seems to use this irony to underline the main point "甚至对即将发生在夏威夷的海啸，也因为远离美国本土，而不大会引起美国媒体太大的兴趣，从而影响他们享受周末烧烤的情绪". Just because you want to watch it 24/7, does not mean others should feel the same or the medium suits your needs. Yes, I did my weekend BBQ too, what the heck is wrong with that? Did you have your lunch and dinner, what was it? Noodles? Is that less disgusting in such an "historical event?" than BBQ, why didn't you skip all the meals since everything in Chile could have been completely leveled?
Talking about the US response, the president went on TV to give condolences and promise to help (US promise to help has always been carried out) the same day, LA urban search and rescue team of hundreds of professionals were on flight readiness a day after, and only a few days back from Haiti, with the knowledge that LA is a high danger area that might need them at any moment too and that's why their team was built with US and CA tax dollars. Secretary Clinton changed trip plan to stop by Chile days later and brought with her material help. Have to disagree with the following "相比一个月前的海地地震，这次更为强烈的地震没有引起美国媒体的迅速关注，显然也有“灾难疲劳”的原因。自然灾难如果频繁发生，人们对其就不再敏感，甚至会无动于衷，正如在战争中人们适应了频繁的轰炸杀戮之后，只要尚未危及自身也会不再害怕，对苦难变得迟钝甚至无动于衷一样。
“事不关己，高高挂起，”笔者心中突然冒出这样一句话来，不知是悲哀，还是嘲讽。不是这样吗？正当圣地亚哥一片火海，遍地狼藉，全国各地上百万所建筑成为无法居住的危房时，夏威夷人却涌向海边，架上摄像机，一副见证盛典或奇迹，抑或一睹也许是百年不遇的巨浪风采。" I don't know how you 事不关己, 低低坐地， I live my life to the best I can, every god-dxxm day, including that eventful day as you see it, and I and my whole family including the history watching kids of 5 and 6, donated money.
This 兴无灭资 has a real clogged view by the ridiculous 无 thinking, because the pen name 笔者心中突然冒出这样一句话来 it sounded so much like 0.5 thinking though.
______________________________
Original author ·兴无灭资·
今天是星期六，醒来第一件事自然是打开床边椅子上的手提电脑，查一下电邮。一打开电邮首页，一副触目惊心的照片跃入眼帘。只见一座坍塌的立交桥下，底朝天地躺着四五辆轿车和载货卡车，就像一具具肢体残缺的尸体，惨不忍睹。再一看标题，才知道自己还在梦乡的时候，智利刚刚发生了一场8.8级的地震。
8.8级，这是一个什么概念？据说在地震仪上，每上升一级，地震强度就增加十倍。最近海地的地震，包括前年的汶川地震，都不超过8度。所以8.8度就意味着十倍的海地和汶川地震，那不早就把太子港和汶川毁灭十次了吗？
但是这个想法很快就被接下来阅读电邮的过程打断，甚至很快就抛掷脑后，不再多想了。直到下楼吃午饭时，打开电视用遥控选台时，才发现一家西班牙语台显然已经对此连续报道多时了。只见主播一会儿连线现场的记者，一会儿连线墨西哥城的一家电视台的演播室，不断在互通情报。同时，电视画面不停地变换着视频和照片，展示智利首都圣地亚哥的受损情况。由于地震发生在海边，所以会引起了太平洋沿岸的海啸。因此，半个小时后，镜头转向了夏威夷。主播甚至连线到当地的居民，向他们询问哪里是否发生了什么状况，并把镜头锁定在夏威夷旅游胜地的一片海滩上。同时，电视屏幕的下方出现了两行文字，说数小时内（solas horas）海啸就会到达夏威夷。笔者离开了电机一会儿，回来就发现文字已经变成了数分钟内（solos minutos）海啸就会到达夏威夷了。这把笔者吓得赶快坐回到沙发上，急切地等待着这一历史时刻的到来。
只是左等右等，看到的只是沙滩岸边的高地和堤坝上聚集的人群越来越大，显然也都像笔者同样急切地想见证一个庄严历史时刻的到来。但是，目光所至，并不见海啸到来的迹象，有的只是几个不畏警告，仍在风口浪尖上弄潮的冲浪者和点缀在蓝白波涛之上的白色风帆。
显然，电视台为了不失去观众，不惜玩弄文字游戏以达到操纵观众的目的。但是笔者对这家电视台放弃既定节目和所有广告，持续跟踪报道正在发生的新闻事件，以满足有关观众知情权的态度，不得不表示欣赏。在等待的过程中，笔者浏览了一下其它电视台的节目，发现除了德国之声正在自己的新闻节目中对此进行一般报道之外，没有一家美国英文电台在报道这个突发新闻。它们的周末电影依旧照常进行。显然，发生在几千英里之外一个南美国家的突发灾难并没没有足够的理由中断美国人惬意的周末。甚至对即将发生在夏威夷的海啸，也因为远离美国本土，而不大会引起美国媒体太大的兴趣，从而影响他们享受周末烧烤的情绪。
结果，笔者只好守住这家西班牙语台，权当练习听力了。由此可见， “血浓于水”的道理，普世皆适。移民美国的智利人和其他南美人，肯定非常关心自己仍在故国的亲人，一定都把眼睛粘在这个频道上了。同时，通过这家电视台呼吁的“帮助智利”捐款活动，已经展开。
相比一个月前的海地地震，这次更为强烈的地震没有引起美国媒体的迅速关注，显然也有“灾难疲劳”的原因。自然灾难如果频繁发生，人们对其就不再敏感，甚至会无动于衷，正如在战争中人们适应了频繁的轰炸杀戮之后，只要尚未危及自身也会不再害怕，对苦难变得迟钝甚至无动于衷一样。
“事不关己，高高挂起，”笔者心中突然冒出这样一句话来，不知是悲哀，还是嘲讽。不是这样吗？正当圣地亚哥一片火海，遍地狼藉，全国各地上百万所建筑成为无法居住的危房时，夏威夷人却涌向海边，架上摄像机，一副见证盛典或奇迹，抑或一睹也许是百年不遇的巨浪风采。
不知什么原因，笔者脑中浮现出《动物世界》中经常出现的镜头：一只老虎或者狮子，一头冲进大群野牛或野鹿丛中，单枪匹马地扑倒其中的一员，然后从容地把它拖到草丛中去从容地享用。而受害者的同类，哪怕成千上万，却只顾东奔西窜，各自落荒而逃。笔者经常天真地假设，它们也无需有意识地团结起来，那样做要求也过高了一些；它们只需依着本能，掉过头来冲着对手做一个简单的stampede，就足以将其碾成灰烬。
同理，人类在面对天灾人祸时，也不必一定要有意识地撇开意识形态的分野和政经利益的博弈，而只需凭着生存的本能，集体地面对危险的挑战。尤其是天灾的危险，对任何人都是一视同仁的，不会因为你是腰缠万贯的富翁就放你一马。于是，为了避免让天灾人祸将人类各个击破，人类哪怕只是为了一己的安危，也应放弃前嫌，忘记各自的种族，宗教，经济和政治的区别和利益，共同应对诸如气候变化，资源匮乏，和环境污染等会致整个人类于死地的难题。
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