IGC

Research

Development of a FACS-based method for selection of mammalian somatic cell knockin clones

Collaborative work with the Epigenetic Mechanisms Lab at the Instituto Gulbenkian Ciência, Oeiras

  • FACS selection knockin clones

  • Mammalian genomes can be site specifically engineered by "targeting" foreign DNA constructs into predefined sites in the genome by recombination. However, such directed integration of DNA is exceeding inefficient and the vast majority of insertions occur elsewhere. Identification and isolation of desired cell clones typically involves weeks of drug selection in culture followed by clone-by-clone PCR genotyping of candidates. Targeting frequencies of 0.1 up to a few percent make this a highly labor intensive process.

    We are developing a FACS-based method that involves a strategy in which cells acquire fluorescence only upon successful targeting to a predefined site. This method provides selective advantage compared to traditional drug selection in that, in principle, no off-target events are detected. Moreover, targeted clones are isolated by FACS within days after introduction of DNA, obviating the need for clonal outgrowth on drug selection. This procedure results in fast and efficient isolation of exceedingly rare genomically engineered somatic cell clones. The challenge we face, and are currently tackling, is the detection and isolation of viable one-in-a-million events.


Sorting Pollen Grain Content from Arabidopsis thaliana

Collaborative work with Jorg Becker's group at the Instituto Gulbenkian Ciência, Oeiras

  • Membrane labeled dilution

  • Arabidopsis thaliana is a small flowering plant widely used as a model organism in plant genetics and development. In particular, pollen tubes of A. thaliana have been used to study cell growth and morphogenesis. Little was known about the expression profile of pollen until the first Arabidopsis pollen grains were isolated by FACS to characterize their transcriptional profile using DNA microarrays (Becker et al, 2003). However, Arabidopsis pollen grains contain two sperm cells (the male gametes) and an accompanying vegetative nucleus, which are likely to contribute differently to the overall gene expression profile observed in pollen. To address the differential genetic contribution of sperm and vegetative nucleus, we are developing a method to isolate sperm cells and vegetative nucleus by FACS, using transgenic A. thaliana pollen expressing different fluorescent proteins.

    We have succeeded in sorting sperm cells in A. thaliana (Borges et al, 2008, Gardner et. al, 2009), and we are now optimizing the procedure to separate simultaneously the vegetative nucleus and sperm cells using trangenic Arabidopsis expressing EGFP in sperm cells and RFP in the vegetative nucleus.

    References:

    1. J. Becker, L. C. Boavida, J. Carneiro, M. Haury and J. A. Feijó (2003) "Transcriptional Profiling of Arabidopsis Tissues Reveals the Unique Characteristics of the Pollen Transcriptome." Plant Physiol. 133 :713-725 [pdf available on request]
    2. F. Borges, G. Gomes, R. Gardner, N. Moreno, S. McCormick, J. A. Feijó, J. D. Becker (2008) "Comparative Transcriptomics of Arabidopsis Sperm Cells" Plant Physiol . 148 : 1168-1181 [pdf available on request]
    3. R. Gardner, F. Borges, T. Lopes, G. Gomes, N. Moreno, J. A. Feijó, J. D. Becker (2009) "Fluorescent-Activated Sperm Cell Sorting in Arabidopsis thaliana." Poster for the FlowCytometryUK 2009 meeting.
    4. R. Gardner, F. Borges, T. Lopes, N. Moreno, J. A. Feijó, J. D. Becker (2010) "Fluorescent-Activated Male Germ Unit Sorting in Arabidopsis thaliana." Poster for the ISAC XXV International Congress: CYTO2010.

Separating X and Y mouse sperm

  • Membrane labeled dilution

  • Based on the need of some of our users to breed only female mice we are optimizing a procedure of X sperm isolation to increase the chance of female progeny. The procedure is divided in four main steps:

    1. Collect mouse sperm
    2. Stain sperm DNA with Hoechst
    3. Sort X sperm (High Hoechst fluorescence)
    4. Fertilize mice by IVF

    Our contribution is to analyze by flow cytometry the changes in fluorescence distribution of cells labeled with a fluorescent derivative of Wheat Germ Agglutinin (WGA-FL) and CFSE in successive division cycles in order to understand the molecular factors and morphodynamic principles underlying the mechanism(s) generating the divison planes in the S. aureus spherical cells.