Genetic modification of mice and of embryonic stem cells (ESCs) is an important source of insight into the functions of vertebrate genes. Current methods commonly used for genetically modifying ESCs are transgene insertion and homologous recombination. Transgene insertion involves integration at a random location in the genome. Homologous recombination can create targeted mutations, but only about 1.5% of ESC clones, on average, correctly integrate the construct and different targeting constructs must be created for each gene.
A complementary approach to generating loss-of-function mutations in ESCs is gene trapping1–3
. Below, we describe the gene trap vectors pGTLxf and pGTLxr, which allow post-insertional modification of the trapped locus using an accompanying technology called Floxin (F
sertion), based on recombination mediated cassette exchange (RMCE).
We show a schematic of mutation, reversion, and modification of a generic, autosomal Your Favorite Gene
) (). A pGTLxf or pGTLxr gene trap in an intron of YFG
results in expression of a YFG-βgeo fusion protein in the gene trap line YFGGt/+
(). To revert this loss-of-function mutation, transiently expressed Cre excises the floxed splice acceptor, leaving a single Lox71 site ()4
. In the absence of the splice acceptor, YFGRev/+
cells lose βgeo
expression and reactivate YFG
The Floxin strategy for reversion and modification of gene trap loci
To permit modification of a gene trap locus, pFloxin vectors contain a Lox66 site () so that Cre-mediated recombination between the pFloxin Lox66 and the genomic Lox71 site of revertant cells results in directional insertion of the pFloxin sequence (). Recombination between Lox66 and Lox71 sites produces one inactive Lox site and one LoxP site (), making this integration irreversible5
Floxin vectors also permit expression of defined sequences at the modified locus (). Although Floxin can facilitate many kinds of gene modifications, we illustrate here the production of carboxy- and amino-terminally tagged alleles of YFG. The pFloxin-YFG-Flag inserted cDNA is spliced so that the inserted sequence is expressed as a fusion with upstream exons from the endogenous promoter. The other Floxin vector, pFloxin-IRES-HA-YFG, contains an IRES element to initiate translation of an amino-terminally tagged version of YFG. The line YFGIRES-HA-YFG/+ expresses full length HA-YFG under the control of the endogenous promoter and the IRES element. The Floxin vectors also include βActin promoters that reactivate βgeo expression and thus permit pharmacological selection of correct insertions ().
RMCE has been shown previously to function robustly in ESCs with varied vector designs6–11
. To date, the BayGenomics and Sanger Institute gene trap efforts have generated 24,149 gene trap cell lines with the pGTLxf and pGTLxr vectors, representing 4,528 individual genes12
. A list of the gene trap alleles is reported here (Supplementary Table 1
), and the cell lines are available to the community through the International Gene Trap Consortium (IGTC) database (www.genetrap.org
Here, we demonstrate modification of eight genomic loci (Sall4
, Sntb2, Pex14
) using gene trap cell lines and the Floxin system. We note that Ofd1Gt
; MGI names for all alleles are provided in Supplementary Table 2
) cells are hemizygous as the Ofd1
gene is X-linked and the E14 gene trap ESCs are male. Consequently, Ofd1Gt
cells do not produce any Ofd1 protein ().
To remove the exogenous splice acceptor, we electroporated gene trap cells with an expression construct for nuclear Cre recombinase. On average, 45% of colonies screened showed proper excision of the splice acceptor (). Revertant cells no longer displayed β-galactosidase activity or neomycin resistance (, Supplementary Fig. 1d
), and reversion caused loss of the βgeo
transcript (Supplementary Fig. 1e
). Genomic PCR and Southern blot confirmed correct excision of the splice acceptor in revertant cells (Supplementary Fig. 1f–g
Efficient reversion of gene trap mutations and Floxin-mediated engineering of new alleles
Using the Floxin strategy, we generated cell lines expressing wild type Ofd1, Suz12, Sall4, Gli2, or Tardbp with carboxy-terminal tags, and lines expressing eGFP
at the Sntb2
, or Tet1
genomic loci. Revertant lines were co-electroporated with the appropriate pFloxin or pFloxin-IRES construct and a nuclear Cre expression construct and selected with neomycin. On average, 86% of resultant ESC colonies contained the correctly integrated pFloxin construct (), and β-galactosidase activity was re-activated (). Genomic PCR and Southern blot confirmed integration in Floxin cell lines (Supplementary Fig. 1g, Supplementary Fig. 2a–c
). These data indicate that Floxin-mediated targeted insertion occurs efficiently and accurately in many different genomic contexts.
Quantitative RT-PCR and immunoblot indicated that revertant alleles are expressed at wild type levels in Sall4Rev/+
( and Supplementary Fig. 2d
). In contrast, Ofd1Rev
cells expressed lower levels of Ofd1 (at the expected size of 110 kD) than wild type cells (). Ofd1 is essential for the formation of the primary cilium13
, and wild type ESCs possess primary cilia whereas Ofd1Gt
cells do not. However, Ofd1Rev
cells possessed primary cilia, indicating that reversion of the gene trap mutation restored gene function (). RT-PCR and sequencing indicated that the reduced Ofd1
expression is attributable to the use of cryptic splice acceptor sites present in the βgeo
cassette (data not shown). Moreover, the βgeo
transcript is detectable by Northern blot in a subset of revertant lines (Supplementary Fig. 1e
). The use of these cryptic splice acceptor sites in certain revertant cell lines suggests that locus-dependent factors affect the restoration of normal expression.
Floxin-inserted alleles were expressed at levels equivalent to the revertant alleles. The tagged alleles for both Sall4Sall4TAP/+
were expressed at wild type levels ( and Supplementary Fig. 2d
). Although Ofd1-Myc protein levels were lower than that of wild type (), production of Ofd1-Myc was also sufficient to support ciliogenesis () and Ofd1-Myc, like endogenous Ofd1, localized to the centrosome (). Suz12-TAP, like endogenous Suz12, localized to the nucleus (), and ESC differentiation led to downregulation of endogenous Suz12 and Suz12-TAP to similar extents ()14
. Together, these data indicate that Floxin cells produce modified proteins that function and are regulated similar to wild type proteins.
In addition to tagged versions of endogenous genes, the Floxin technology can insert exogenous DNA into loci. Sntb2IRESeGFP/+
lines expressed eGFP under the control of endogenous regulatory elements (Supplementary Fig. 2e
The presence of vector sequences may affect normal gene expression15
. Therefore, we included Frt sites in the gene trap and Floxin vectors to allow for Flp-mediated removal of the βActin
promoter and βgeo
cassette (Supplementary Fig. 3a
). We electroporated Ofd1Ofd1myc
cells with a FLPo expression construct16
and identified cells in which the βActin
cassette was successfully excised by loss of β-galactosidase activity (). Genomic PCR verified correct excision (Supplementary Fig. 3c
). Removal of vector sequences increased the expression of Ofd1-Myc (Supplementary Fig. 3d
To assess whether the genetic manipulations associated with gene trapping, reversion, and the Floxin process affected ESCs, we performed karyotyping and evaluated pluripotency by three methods. The five Floxin lines evaluated showed normal euploid karyotypes (data not shown). All Floxin lines had normal ES colony and cell morphology (Supplementary Fig. 4a
). Additionally, Floxin lines had expression levels similar to wild type ESCs for three regulators of pluripotency, Oct4
(Supplementary Fig. 4b
), and could differentiate into cell types that express Fgf5
, markers of embryonic ectoderm, endoderm and mesoderm, respectively (Supplementary Fig. 4c
). While these assays suggest that the Floxin process does not adversely affect ESC pluripotency, germline competency of Floxin cells has not been systematically evaluated. Other studies have shown that three genetic manipulations do not necessarily limit germline transmission7, 17
A detailed understanding of gene function requires the generation of a range of alleles. The Floxin strategy described here allows for high throughput modification of ESC loci harboring insertions of the pGTLxf or pGTLxr gene trap vectors. The Floxin system allows for Cre-mediated reversion of the gene trap mutation and subsequent insertion of new DNA of interest into the genomic locus. Genes of interest are cloned with standard molecular biology techniques into pFloxin shuttle vectors. With each manipulation, the presence or absence of βgeo expression assists in the selection or identification of the desired cells.
We have demonstrated the generation of tagged or reporter alleles at eight loci, and shown how the technology can be used to model a human genetic disease in ESCs, study protein localization, and report on the dynamics of protein expression (Supplementary Table 3
). The gene trap allele can also be converted into a wide variety of other tailored alleles, such as missense, deletion, and domain swap alleles, and Floxin can be used to insert non-homologous DNA sequences into endogenous loci. This approach could be utilized for generating alleles expressing other exogenous proteins such as Cre, rtTA, ΦC31 integrase, Alkaline phosphatase, or Diptheria toxin under the control of tissue- or cell type-specific promoters.
The Floxin strategy has several advantages over previously described approaches for recombinase-mediated insertion of exogenous DNA elements6–8, 10
. First, reversion allows for confirmation that observed cellular phenotypes are due to the gene trap mutation, as demonstrated for the role of Ofd1
in ciliogenesis. Second, inserted DNA sequences can be expressed either as a direct fusion to upstream exons, or as a separate cistron. Third, Frt sites allow for removal of the βActin
cassette, abrogating interference from vector prokaryotic sequences. Lastly, the Floxin technology is compatible with the extensive collection of 24,149 characterized and validated gene trap lines available to the community.
The Floxin strategy of reversion and new DNA insertion are both highly efficient and reproducible at a variety of loci. By avoiding the most laborious aspects of traditional gene replacement strategies, the Floxin system allows new alleles to be engineered with minimal effort.