gene trap mutagenesis

To identify growth factor regulated genes, we are using gene traps in embryonic stem (ES) cells.  In this approach, a promoterless reporter gene (for instance encoding βgalactosidase, or βgeo) is introduced in ES cells.  Selection for expression of the gene requires transcription from a cellular promoter, and consequently a mutation in a cellular gene, and the activity of the tagged gene can be followed by staining for βgalactosidase activity.  Detailed description of methods used for gene trap mutagenesis may be found in Friedrich, G., and Soriano, P. (1993) and Chen, W.V., and Soriano, P(2003).  Large scale sequencing of ES cell clones was conducted and sequence tags have been deposited to NCBI's dbGSS.  Sequence searches for genes that we have trapped can be performed by running a BLAST search of the dbGSS database.  A list of the genes we have trapped can be found in our gene trap database.

Vector design

The basic gene trap vectors we have used include a reporter gene downstream of a splice acceptor sequence (Figure 1).  They are therefore designed to function when inserted in an intron.  The gene trap cassette is inserted in reverse orientation in a retroviral vector, and vectors and mutant lines derived from our screens are referred to as ROSA (Reverse-Orientation-Splice-Acceptor).  Retroviruses insert as a single copy per locus, with no rearrangement of flanking sequences.  They have a preference for insertions at the 5' end of genes, often upstream of the initiator ATG, and the splice acceptor sequence we use does not appear to be bypassed.  As a result, the majority of the mutations generated using our gene trap vectors are predicted to lead to null alleles.  This has in fact been verified in all of the insertions we have analyzed to date at a molecular level.

Figure 1: Gene Trap Design

Phenotypic analysis

Among 70 mutations that have been transmitted through the germ line (all called ROSA), about one third result in a recessive phenotype, either affecting embryonic development at different stages, or the adult.

Lines                Vector            Screen            Phenotype                    Gene                            Strain

SA βgeo 1-5         SA βgeo              Random                      2/  5 Emb. lethal                Sec8 ("Spock")                         Saβgeo 4

ROSA 1-29           ROSA βgeo        Random                      7/29 Emb. lethal                TEF1                                         ROSA 5

                                                                                            1/29 Male sterile                Transmembrane                       ROSA 22

                                                                                                                                       Nuclear RNA                            ROSA 26

ROSA 30-39         FUSA βgeo        Random                       7/10 Emb. lethal

ROSA 40-45         ROSA βgal         Induction(EB)              1/  7 Emb. lethal

                                                                                            1/  7 Male sterile                 Bcl-W                                       ROSA 41

ROSA 46-52         ROSA βgal         Induction                     1/  7 Emb. lethal                 CD98                                        ROSA 49

                                                        (TGFβ)

ROSA 53-55         ROSA βgalCre    Random                     1/  3 Emb. lethal                 Shroom                                    ROSA 53

ROSA 56-61         ROSA βgeo*       Sequence                   1/  3 Growth retarded         HMG Box                                 ROSA 56

                                                                                            1/  3 Emb. lethal                 CTBP2                                     ROSA 61

ROSA 62-65         ROSA βgeo*       Induction (RA)            1/  4 Neuropathy                 βIV-spectrin                             ROSA 62

                                                                                            1/  4 Male sterile                 E-MAP 115                              ROSA 63

ROSA 66-70         ROSA βgal         Induction (serum)        1/  5 Growth retarded         EFII                                          ROSA 67

                                                                                                                                       AA109197                                ROSA 68

ROSA 71              ROSAFARY      Gene Trap Array           Emb. lethal                       Strap                                         ROSA 71

ROSA 72-83        ROSAFARY     Gene Trap Array          Neonatal lethal                     Zfand5, BC058969, Myo1e      ROSA72-83

                                                                                          postnatal phenotypes           Arid5b, BC055757, Schip1

                                                                                                                                       Sgpl1, Tiparp, Axud1,

                                                                                                                                       Mzf6d, Plekha1, r-Txnip

Induction trapping

To identify and mutate growth factor or retinoid regulated genes, we initially performed induction trapping by monitoring differential expression of the reporter gene, in ES cells or their differentiated derivatives.  In some cases, such as ROSA49, we have replica plated ES cells infected with ROSAβgal and tested for differential lacZ activity upon exposure to exogenous factors.  In other instances, such as ROSA62 or ROSA63, we have used a modified version of βgeo for the same purpose.  βgeo encodes a βgalactosidase-neomycin phosphotransferase fusion protein and carries a mutation in the neo moiety that reduces its activity.  This mutation has been corrected in βgeo*.  As a result, induction trapping using ROSAβgeo* is more efficient as all neo resistant colonies are gene trap events yet only a fraction (~60%) exhibit βgalactosidase activity.  We have also designed a novel vector system to isolate inducible gene traps by flow sorting (Medico et al., 2001).

It has been known for many years that activation of growth factor signaling pathways leads to the expression of multiple Immediate Early Genes (IEGs), yet their role in specifying particular growth factor responses remained controversial as similar IEGs are often engaged following induction of multiple signaling pathways. We have used gene trap-coupled microarray analysis, using a modified gene trap vector system in which 3' RACE products from mutated genes are spotted on arrays (Chen et al., 2004), to identify targets of PDGF signaling, and their function in PDGF regulated processes.  Mutations in these genes lead to a high frequency of phenotypes that affect the same cell types and processes as those controlled by PDGF signaling (Schmahl et al., 2007). These results suggest that these genes form a network that control specific processes downstream of PDGF signaling.  We are also using gene traps to perform gain of function or suppressor genetics on sensitized backgrounds.

Results from large scale sequencing of ES cell clones and sequence tags have been deposited to NCBI's dbGSS.  Sequences searches can be performed by running a BLAST search of the dbGSS database.  A list of the genes we have trapped can be found in our gene trap database.  A number of laboratories performing gene trap mutagenesis, including ours, have formed a gene trap consortium. Through the gene trap consortium, cell lines are made available to investigators at non-profit institutions for a modest fee (to recover costs).  These cell lines may be ordered from the MMRRC.  You can also search for our clones in the MMRRC catalog.