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  • Department of Embryology Centennial Symposium | Department of Embryology
    Embryology and to commemorate this occasion we re planning a symposium to be held September 26th and 27th of this year The first day of the symposium is devoted to honoring the exceptionally productive history of the department highlighting aspects of the department that have led to its success Speakers for the first day will feature present former department members and will include Gerry Rubin Jennifer Lippincott Schwartz Steven McKnight

    Original URL path: http://emb.carnegiescience.edu/event/department-embryology-centennial-symposium (2013-06-13)
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  • Embryology Building Dedication Speech to Carnegie Trustees | Department of Embryology
    risks with institutional funds when the very conservative government funding processes decline to fund an interesting idea Leadership that is prepared to rejoice in success but is also prepared for failure for finding even after years of work and a good pile of money that there is after all no story Special admiration is due to the Carnegie board of trustees and its chairs for decades of such leadership I cannot begin to convey how much help and support I had from chairmen Dick Heckert and Tom Urban during my time as president And I know that Mike Gellert is similarly engaged with Dick Meserve So once upon a time in 1914 to give the time a date Franklin Mall convinced the trustees that it was time to learn more about human embryonic development It was the birth of this department He brought to Carnegie his own collection of human embryos obtained from spontaneous abortions and cadavers The embryos amassed and studied by Mall and those who followed was the largest collection of human embryo specimens in the world Now housed at the National Museum of Health and Medicine it remains a resource for those studying human embryology The 23 stages of early human development defined in this department early in the last century remain the framework for the study of human embryos Stage 1 is the fertilized egg and at stage 23 about 8 weeks later the embryo has many features characteristic of a human Stage 3 is the now well known blastocyst the source of human embryonic stem cells But this morphological history of early human development like most great scientific achievements raised more questions than it answered Scientists and physicians wanted to know more about the events leading up to ovulation the process of fertilization and then most mysterious how the single fertilized cell could on its own multiply and form arms fingers legs toes backbone ears eyes and all the other structures visible by Stage 23 So once upon a time in 1925 department director George Streeter convinced President John Merriam and the trustees that the very large expense entailed in establishing a living breeding monkey colony was worth it This amounted to 10 000 or 18 percent of the department budget in 1926 Monkeys were a new kind of model organism and challenging to maintain compared to the flies and rodents already being investigated A great number of the fundamental aspects of human reproduction that are now common knowledge were originally revealed by work with the Carnegie monkey colony including knowledge of the very earliest embryos Primates were essential to this because only primates have a menstrual cycle associated with ovulation allowing among other things the precise determination of the age and morphology of the earliest embryos My own first introduction to the Carnegie Institution came in 1951 my junior year in college when my wonderful embryology professor Robert Enders assigned as a textbook a monograph entitled Embryology of the Rhesus Monkey Collected papers from

    Original URL path: http://emb.carnegiescience.edu/embryology-building-dedication-speech-carnegie-trustees (2013-06-13)
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  • Postdoctoral Training Program | Department of Embryology
    exceptional record of Carnegie trained researchers Above all the Carnegie style is ideally suited to stimulate creative ventures that open up the new opportunities needed for a successful postdoc Moreover postdocs at Carnegie operate within a training environment that maximizes the acquisition of the skills and knowledge that will be useful in their future endeavors The department as a whole maintains a broad scientific base in the biological sciences through numerous departmental seminars symposia journal clubs and progress reports that maximize scientific exchanges and interactions There is a strong emphasis on enhancing the ability of postdocs to effectively present their research in both written and oral form Finally with its location in the thriving Baltimore Washington metropolitan area and on the Johns Hopkins University campus the department is well positioned for diverse scientific interactions and collaborations The Carnegie Collaborative Fellowship The Carnegie Collaborative Fellowship program aims to identify on a yearly basis one or two exceptionally creative graduate students who are capable of thinking outside the mainstream and to tailor a postdoctoral experience that will allow them to pursue a research program that exceeds the boundaries of any single laboratory For example a fellow might initiate research on a process

    Original URL path: http://emb.carnegiescience.edu/postdoctoral-training-program (2013-06-13)
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  • Staff Associate Program | Department of Embryology
    merits to be revealed Staff Associates are independent junior faculty members who hold non renewable faculty level appointments for up to five years They may be appointed in lieu of or after completing a regular postdoctoral fellowship It is a time for these young scientists to build a research program with no distractions Staff Associates work at the bench in their own laboratory space often with the help of a technician or summer student While pursuing their research goals Staff Associates remain closely connected to the Department attending seminars and lab meetings with faculty groups of their choice Because the Staff Associate program has succeeded in nurturing many exceptional scientists the number of Staff Associate positions has been increased in recent years Former Staff Associates Name Years Current Title Position Pat Gearhart 1979 1981 National Institute on Aging National Institutes of Health Steve McKnight 1980 1982 Chairman Biochemistry University of Texas Southwestern Medical Center Rick Rotundo 1982 1984 Professor Cell Biology Anatomy University of Miami Sondra Lazarowitz 1983 1990 Professor Plant Pathology Cornell University Martin Snider 1983 1985 Professor Biochemistry Case Western Reserve David Schwartz 1985 1989 Professor Chemistry and Genetics University of Wisconsin Madison Phil Beachy 1986 1988 Professor Developmental Biology Stanford University School of Medicine and Investigator Howard Hughes Medical Institute Andy Fire 1986 1989 Scientific Staff Departments of Pathology and Genetics Stanford Medical School Se Jin Lee 1989 1991 Professor Molecular Biology and Genetics Johns Hopkins University School of Medicine Denise Montell 1989 1992 Professor Molecular Cellular and Developmental Biology University of California Santa Barbara Nipam Patel 1991 1995 Professor Integrative Biology University of California Berkeley Catherine Thompson 1992 1996 Principal Research Scientist Pfizer Inc Susan Dymecki 1993 1998 Professor Genetics Harvard Medical School Pernille Rørth 1995 1998 Research Director Institute of Molecular and Cell Biology A

    Original URL path: http://emb.carnegiescience.edu/staff-associate-program (2013-06-13)
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  • Tan Lab | Department of Embryology
    Perl Thursdays May 30 2013 refining session gt1kbexon v2 pl May 2 2013 Hello world filter for exons on strand 1 kb mouse gtf gt1kbexon pl Nitty Gritty Workflows June 4 2013 Will Yarosh searching for DNA structural variants using igvtools and awk March 12 2013 Gaëlle Talhouarne motif finding detecting circles January 29 2013 Mike Harris heterogeneous samples calculating enrichment January 4 2013 Eric Mills count tables variance estimation

    Original URL path: http://emb.carnegiescience.edu/labs/frederick-tan/teaching (2013-06-13)
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  • Tan Lab | Department of Embryology
    intergenic counts 5 3 bias preseq Estimates and predicts library complexity CummeRbund R package to visualize Cufflinks data SeqMonk Java package to visualize mapped reads interactive scatter MA plots DAVID Web based GO analysis with extensive gene ID mapping blast2go Java program to assign GO terms to novel genes goseq R package for GO analysis that normalizes for gene length BEDTools HTSeq SAMtools Picard BamTools FASTX Toolkit SRA Toolkit bam2fastq BALTIMORE AREA SEQUENCING Next Generation Sequencing Center CRB II 131 GRCF High Throughput Sequencing Center Blalock 1005 JHMI Deep Sequencing Microarray Core Broadway 351 Center for Computational Genomics USEFUL REFERENCES SEQanswers removing duplicates TopHat MAPQ cuffmerge vs cuffcompare Biostar Galaxy s mailing list search Illumina s video of how image data is acquired and initially analyzed Synthetic spike in standards for RNA seq experiments pmid 21816910 Transcription or translation pmid 21593866 Rafalab Videos TRAINING AND TUTORIALS mRNA Seq using TopHat and Cufflinks pmid 22383036 small RNA sequencing experiments pmid 21356093 Short Courses and Workshops JHMI CCG CGRL UC Berkeley Bioinformatics Training Program UC Davis Berkeley Seq Bioinformatics Organization Educational Services Stephen Turner s list of bioinformatics workshops and resources Unix and Perl Primer for Biologists by Keith Bradnam and Ian

    Original URL path: http://emb.carnegiescience.edu/labs/frederick-tan/bioinformatics (2013-06-13)
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  • Graduate Student Training Program | Department of Embryology
    students since its inception in 1914 Graduate training is possible in any of the seven laboratories within the department Although each lab has a separate scientific focus the department as a whole maintains a broad scientific base in biological sciences through numerous scientific exchanges and interactions departmental seminars and symposia journal clubs and progress reports These and more informal interactions provide students and postdocs with broad training in biological sciences With its location on the Johns Hopkins University Arts and Sciences campus and scientific affiliations with the Carnegie Institution and Johns Hopkins University the department also provides extensive opportunities for scientific collaboration Graduate Study in Biology Johns Hopkins University Each principal investigator at Carnegie holds an adjunct faculty appointment in the Department of Biology at Johns Hopkins Graduate students in biology at Johns Hopkins can thus carry out thesis research in any of the laboratories in the Department of Embryology The Biology and Embryology departments are situated approximately 1 4 mile apart and are both on the tree lined Arts and Sciences campus of Johns Hopkins University Biology graduate students who choose to carry out their thesis research at Carnegie remain full members of the Biology department and participate fully

    Original URL path: http://emb.carnegiescience.edu/graduate-student-training-program (2013-06-13)
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  • The Drosophila Gateway™ Vector Collection | Department of Embryology
    in EGFP and ECFP but can be distinguished based on size relative to the other epitopes Table 4 Restriction enzymes with sites in the listed epitope for use in verifying destination vectors FAQs I strongly recommend thoroughly reading the Gateway Technology manual from Invitrogen The information we ve provided here is only intended to demonstrate how we routinely use the Gateway system in our lab How do I flank my ORF with attL1 and attL2 recombination sites Several options are available to produce an Entry clone with your ORF positioned in the proper reading frame We routinely amplify the desired ORF and clone it into an Entry vector by a topoisomerase catalyzed reaction pENTR D TOPO Cloning Kit Invitrogen catalog K240020 Primers are designed with the sequence CACC on the 5 end of the 5 primer followed by the gene specific sequence in the first reading frame The 5 end of the 3 primer should end with a complete codon Figure 3 If you plan on making C terminal fusions your PCR product must include an ATG typically immediately after the CACC tail and must not contain a stop codon Note that the CACC sequence is a consensus Kozak site and will be included in the final clone The resulting construct can also be used to make N terminal fusions but will contain a short tail at the C terminus which can be detrimental to some proteins Figure 3 PCR product design for TOPO cloning The resulting Entry clone will have the sequence shown in figure 4 where the AAA AAA and TAC AAA sequences represent the core of the attL1 and attL2 recombination sites For the PCR reaction we routinely use a proofreading polymerase such as Pfx Invitrogen catalog 11708 021 to amplify from a cDNA and have observed a PCR induced error rate of 1 15 000 bases after cloning from a 25 cycle amplification In our hands we need to use a much lower concentration of MgSO4 in the PCR reaction 0 25 mM than recommended by Invitrogen in order to get a PCR product We have found that the TOPO cloning reaction isn t as efficient or directional as claimed by Invitrogen We recover a median of 42 colonies range 17 356 of which a median of 25 range 8 56 contain the PCR product cloned in the correct orientation However the colonies can be rapidly screened by colony PCR using a vector specific and ORF specific primer Afterwards we miniprep and sequence verify a single clone which becomes our master Entry clone for recombination into the desired Destination vectors Entry clones can also be produced by including attB1 and attB2 sequences in the PCR primers and using a BP recombination reaction to recombine the attB sites with attP sites in a donor vector resulting in an entry clone with your ORF flanked by attL1 attL2 recombination sites This strategy can also be used to directly recombine your ORF into a Destination vector by sequentially carrying out a BP followed by a LR recombination reaction as described in the Gateway manual However we prefer to use TOPO cloning to avoid ordering primers that include the 29 bp attB1 attB2 sequences We also prefer sequence verifying the Entry clone which can then be used with many different Destination vectors Figure 4 Entry clone sequence after cloning into pENTR D TOPO How do I ensure that my ORF will be in the proper reading frame after recombining into a destination vector As long as your ORF is in frame with the AAA AAA sequence at the 5 end and the TAC AAA sequence at the 3 end e g Figure 4 the resulting clone will be in frame with either N terminal or C terminal epitope tags What materials do I need to use the Drosophila Gateway Vector Collection To make an Entry clone containing your ORF using pENTR D TOPO pENTR D TOPO Cloning Kit Invitrogen catalog K240020 20 reactions 20 rxn which includes TOP10 competent cells cDNA of your favorite gene DGC clone or equivalent 20 per clone shipping from BACPAC PCR primers to amplify the ORF 12 pair Proofreading polymerase e g Pfx Invitrogen catalog 11708 021 or equivalent 0 80 rxn LB agar plates containing 50 ug ml kanamycin Primers to sequence verify longer ORFs 1000 bp after cloning We occasionally perform half reactions of the TOPO cloning step to reduce costs although this results in a 3 ul reaction volume which can be hard to manage To recombine an Entry clone with a Destination clone miniprep DNA of an Entry clone containing your ORF miniprep DNA of the desired Destination vector Gateway LR Clonase Enzyme Mix Invitrogen catalog 11791 019 20 reactions or 11791 043 100 reactions 12 50 to 14 75 rxn Library Efficiency DH5alpha competent cells Invitrogen catalog 18263 012 or equivalent 2 50 20 ul LB agar plates containing 100 ug ml ampicillin We routinely perform half reactions 10 ul total volume of the LR Clonase step to reduce costs and recently had success with quarter reactions 5 ul total volume Which destination vector should I use The beauty of this system is that you can easily make several constructs for different purposes For example we initially examine protein localization in Drosophila KC cells using pHGW and pHWG Hsp70 promoter GFP fusions at either end This way we can test which end of the protein better tolerates the epitope tag Proteins that we want to further analyze in vivo are then swapped into pTGW or pTWG The HA Myc and FLAG epitope tags are all useful for immunoprecipitations in culture or in vivo we have only used the HA tag in tissue culture experiments We are just beginning to use the Venus and mRFP constructs The Venus tag EYFP should be more versatile than EGFP it is brighter works with standard GFP filter sets and can be easily spectrally separated from ECFP for double labeling experiments Consequently we anticipate that

    Original URL path: http://emb.carnegiescience.edu/labs/murphy/Gateway%20vectors.html (2013-06-13)
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