Understanding how molecules act together to build complex systems from the sub-cellular to organismal level is dependent on our ability to uncover the dynamics of their localisation and physical interactions in vivo.
One challenge of making labeled transgenes is tagging them while maintaining their endogenous regulation. A typical way to do this is to use cDNAs, but these lack the relevant cis regulatory sequences and introns. One way to preserve the regulatory structure of genes is to use bacterial artificial chromosomes, or BACs. These can be upwards of 200KB and, for most genes, will contain the bulk of the cis regulatory sequences. However, modification of BACs proves to be challenging. One cannot use conventional restriction digests to work with them because of their large size.
A key step forward in this regard was the development of bacterial-based recombineering, in which BACs are modified inside bacteria by viral enzymes (Zhang et al, Nature Genetics 1998). The use of recombineering in large scale has been a Dresden success story, fostering cross-campus and cross-institute collaborations between our lab, the groups of Francis Stewart (BIOTEC), Frank Buchholz (TUD), research group leaders Elisabeth Knust, and Pavel Tomancak (MPI-CBG), and Mihail Sarov, who runs the TransgenOmics facility.
TransgeneOmics in the Hyman Lab
In the Hyman lab, we are specifically interested in using TransgeneOmics to analyze proteins’ functions during cell division. Towards this goal, our staff scientist Ina Poser, in conjunction with Frank Buchholz and others, established a procedure to transfect BAC transgenes into HeLa cells and mouse embryonic stem cells (Poser et al, Nature Methods, 2008, and Kittler et al, PNAS, 2005). Their procedure creates stable cell lines for studying the function of thousands of tagged proteins in mammalian tissue.
We have used transgeneomic techniques to tag and make transformed cell pools for over 1000 genes that have been implicated in cell division by RNAi screens. Importantly, all tagged proteins are expressed under their own regulatory sequences. This library is used for follow-up experiments with mass spectrometry, RNAi, and localization, and was integral for our contributions to the successful MitoCheck project. The 5-year MitoCheck consortium systematically screened all 22,000 human genes by RNAi and found that ~600 play a role in mitosis (Hutchins et al, Science 2010; Neumann et al, Nature 2010).
The Hyman lab’s expert TransgeneOmics team works together to create thousands of highly specialised transgenic cell lines. Genes are tagged in any number of ways, with a variety of fluorescent tags and resistance selection markers, in order to perform localization and co-localization analysis. We also create transgenes with specific point mutations that mimic naturally occurring mutants in order to better understand the direct effect such mutations have on protein function.
In combination with counter selection to introduce point mutants and make the genes RNAi resistant, TransgeneOmics provides a very powerful way to perform structure-function studies in human cells. Such structure-function studies in vivo, which are based on single molecule results in vitro, are extremely powerful methods to test models for mitotic spindle assembly and form the core of MitoSys, a new EU-integrated project coordinated by Jan Michael Peters in Vienna. The MitoSys project includes modelers, biophysicists, and biochemists from all over Europe with an interest in modeling mitosis. We are working within this network to model mechanisms of mitotic spindle assembly.
We are continually expanding our lab’s library of tagged BACs and intend to reach 10,000 human genes in the near future, making these lines available for distribution. Currently, stable transformed cell lines made in the Hyman lab have been sent to over 150 labs in 26 different countries.
Obtaining Cell Lines
View our BAC and Cell Line Database, BaCe Bank, to search our stable HeLa BAC lines. If you are interested in obtaining any of our TransgeneOmics HeLa cell lines, please contact Ina Poser for more information.
In 2008 we began a collaboration with the Fraunhofer Institute to develop an automated system for culturing and handling our TransgeneOmics cell lines. This cooperative project is called Autranomics. Watch the video below to see our cell culturing robot in action!
Our TransgeneOmics team is part of the following research consortia, past and present. Click on each logo to learn more.
Members of the Hyman lab working on TransgeneOmics are:
Annett Dümmler, Susanne Ernst, Marit Leuschner, Andrei Poznaikovsky, Andrea Ssykor, Yusuke Toyoda
Key Publications (for a complete list, visit our Publications Page)
- Maliga Z, Junqueira M, Toyoda Y, Ettinger A, Mora-Bermúdez F, Klemm RW, Vasilj A, Guhr E, Ibarlucea-Benitez I, Poser I, Bonifacio E, Huttner WB, Shevchenko A, Hyman AA. A genomic toolkit to investigate kinesin and myosin motor function in cells.
Nat Cell Biol. 2013 Mar;15(3):325-34. [PubMed] [PDF]
- Bird AW, Erler A, Fu J, Hériché JK, Maresca M, Zhang Y, Hyman AA, Stewart AF. High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes
Nat Methods. 2011 Dec 4;9(1):103-9. [PubMed]
- Neumann B, Walter T, Hériché JK, Bulkescher J, Erfle H, Conrad C, Rogers P, Poser I, Held M, Liebel U, Cetin C, Sieckmann F, Pau G, Kabbe R, Wünsche A, Satagopam V, Schmitz MH, Chapuis C, Gerlich DW, Schneider R, Eils R, Huber W, Peters JM, Hyman AA, Durbin R, Pepperkok R, Ellenberg J. Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes.
Nature. 2010 Apr 1;464(7289):721-7. [PubMed]
- Hutchins JR, Toyoda Y, Hegemann B, Poser I, Hériché JK, Sykora MM, Augsburg M, Hudecz O, Buschhorn BA, Bulkescher J, Conrad C, Comartin D, Schleiffer A, Sarov M, Pozniakovsky A, Slabicki MM, Schloissnig S, Steinmacher I, Leuschner M, Ssykor A, Lawo S, Pelletier L, Stark H, Nasmyth K, Ellenberg J, Durbin R, Buchholz F, Mechtler K, Hyman AA, Peters JM. Systematic analysis of human protein complexes identifies chromosome segregation proteins.
Science. 2010 Apr 30;328(5978):593-9. (Epub 2010 Apr 1) [PubMed]
- Poser I, Sarov M, Hutchins JR, Hériché JK, Toyoda Y, Pozniakovsky A, Weigl D, Nitzsche A, Hegemann B, Bird AW, Pelletier L, Kittler R, Hua S, Naumann R, Augsburg M, Sykora MM, Hofemeister H, Zhang Y, Nasmyth K, White KP, Dietzel S, Mechtler K, Durbin R, Stewart AF, Peters JM, Buchholz F, Hyman AA. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.
Nat Methods. 2008 May;5(5):409-15. [PubMed]
- Kittler R, Pelletier L, Heninger AK, Slabicki M, Theis M, Miroslaw L, Poser I, Lawo S, Grabner H, Kozak K, Wagner J, Surendranath V, Richter C, Bowen W, Jackson AL, Habermann B, Hyman AA, Buchholz F. Genome-scale RNAi profiling of cell division in human tissue culture cells.
Nat Cell Biol. 2007 Dec;9(12):1401-12. [PubMed]
- Kittler R, Pelletier L, Ma C, Poser I, Fischer S, Hyman AA, Buchholz F. RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells.
Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2396-401. [PubMed]