MICE, a program to track and monitor animals in animal facilities

BACKGROUND: A growing number of laboratories are using the mouse as a model system in developmental biology as well as in molecular biology. Surprisingly, most of these laboratories do not have reliable computerized systems to track these animals, and the few commercial solutions available are expensive. We thus developed MICE (Mouse Information and Classification Entity), a program aimed at facilitating the monitoring of animals in animal facilities. RESULTS: This program consists of a virtual facility in which scientists can perform all the tasks done in the real world (i.e., receiving animals, breeding them, preparing cage labels, etc.). Recording of each animal (birth date, cage number, ID number, tail analysis number, parents, genetic status, genetic background, etc.) enables reliable tracking. According to any parameter of interest, animals can then be identified, grouped, sorted, moved, and so forth. Crossings are automatically processed by the program. For example, new genetic backgrounds, generation number, and anticipated due dates are determined. The program also reminds the user when new births are expected and entering newborn animals only requires a few clicks. The genealogy of each animal can be determined in two different ways, one being the visualization of a genealogical tree from which information of ancestors can be retrieved. CONCLUSION: This standalone program, that will be distributed free of charge to academic laboratories requesting a license, represents a new and valuable tool for all animal facility users, and permits simple and reliable tracking and retrieving of animals

REtools: A laboratory program for restriction enzyme work: enzyme selection and reaction condition assistance

BACKGROUND: Restriction enzymes are one of the everyday tools used in molecular biology. The continuously expanding panel of known restriction enzymes (several thousands) renders their optimal use virtually impossible without computerized assistance. Several manufacturers propose on-line sites that assist scientists in their restriction enzyme work, however, none of these sites meet all the actual needs of laboratory workers, and they do not take into account the enzymes actually present in one's own laboratory. RESULTS: Using FileMaker Pro, we developed a stand-alone application which can run on both PCs and Macintoshes. We called it REtools, for Restriction Enzyme tools. This program, which references all currently known enzymes (>3500), permits the creation and update of a personalized list of restriction enzymes actually available in one's own laboratory. Upon opening the program, scientists will be presented with a user friendly interface that will direct them to different menus, each one corresponding to different situations that restriction enzyme users commonly encounter. We particularly emphasized the ease of use to make REtools a solution that laboratory members would actually want to use. CONCLUSION: REtools, a user friendly and easily customized program to organize any laboratory enzyme stock, brings a software solution that will make restriction enzyme use and reaction condition determination straightforward and efficient. The usually unexplored potential of isoschizomers also becomes accessible to all, since REtools proposes all possible enzymes similar to the one(s) chosen by the user. Finally, many of the commonly overlooked subtleties of restriction enzyme work, such as methylation requirement, unusual reaction conditions, or the number of flanking bases required for cleavage, are automatically provided by REtools

A new generation of pPRIG-based retroviral vectors

<p>Abstract</p> <p>Background</p> <p>Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).</p> <p>Results</p> <p>The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (« modular » PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (« single color/resistance » PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (« dual color/selection » PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells.</p> <p>Conclusion</p> <p>These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (« modular » PRIGs), for multiple transductions and fluorescence analyses of transduced cells (« single color/resistance » PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (« dual color/selection » PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.</p

Development of a new bicistronic retroviral vector with strong IRES activity

BACKGROUND: Internal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies. RESULTS: We observed up to a 10 fold difference in IRES-controlled expression from distinct bicistronic expression vectors harboring the same apparent IRES sequences. We show that the insertion of a HindIII site, in place of the initiating AUG codon of the wild type EMCV IRES, is responsible for the dramatic loss of expression from the second cistron, whereas expression from the first cistron remains unaffected. Thus, while the replacement of the authentic viral initiating AUG by a HindIII site results in the theoretical usage of the initiation codon of the HindIII-subcloned cDNA, the subsequent drop of expression dramatically diminishes the interest of the bicistronic structure. Indeed, insertion of the HindIII site has such a negative effect on IRES function that detection of the IRES-controlled product can be difficult, and sometimes even below the levels of detection. It is striking to observe that this deleterious modification is widely found in available IRES-containing vectors, including commercial ones, despite early reports in the literature stating the importance of the integrity of the initiation codon for optimal IRES function. CONCLUSION: From these observations, we engineered a new vector family, pPRIG, which respects the EMCV IRES structure, and permits easy cloning, tagging, sequencing, and expression of any cDNA in the first cistron, while keeping a high level of expression from its IRES-dependent second cistron (here encoding eGFP)

Transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein by Ets, Rel/NFKB, STAT and API transcription factor families

Transcription factors play an essential role in determining the fate of a cell by affecting the expression of target genes involved in proliferation, in differentiation and in programmed cell death. Under certain conditions, some of these factors are capable of deregulating expression of genes involved in the cell cycle andlor in programmed cell death resulting in uncontrolled proliferation of the cell. The focus of this review is on the transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein. Since 1999, severa1 papers have implicated members of the Ets, R~~/NFKSBT, N and AP-1 families as transcription factors regulating bcl-x expression. A specific emphasis of these different transcription factor families on bcl-x regulation in hematopoietic cells is discussed

Virtual Nitrogen Tank to Monitor Frozen Cell Stocks

We developed a program to facilitate the monitoring of biological samples (cell lines, sera, etc.) that are stored in liquid nitrogen containers. The program consists of a “virtual” container in which scientists can store their samples and a program that records the location of each sample, cell characteristics, storage dates, names of the manipulators and much more. Additional comments and a photograph can be associated with each vial, allowing for reliable tracking of samples. Vials can then be identified according to any parameter of interest to the scientist, including associated comments. Once identified, the program visually presents the location of these vials, which simplifies retrieving them from the real container. The program records the thawing of vials, along with the date and the name of the operator. Any academic laboratory requesting this standalone program will be granted a free license for its use

Development of a new bicistronic retroviral vector with strong IRES activity-5

<p><b>Copyright information:</b></p><p>Taken from "Development of a new bicistronic retroviral vector with strong IRES activity"</p><p>BMC Biotechnology 2006;6():4-4.</p><p>Published online 12 Jan 2006</p><p>PMCID:PMC1373653.</p><p>Copyright © 2006 Martin et al; licensee BioMed Central Ltd.</p> from the indicated vectors were loaded in duplicate, ran on SDS-PAGE, and analyzed with an anti GFP serum (left panel) and an anti-HA serum (right panel)