Use of a cryptic 3′ SS in NPT is predicted when U3NeoSV1 inserts in an intron of an expressed gene. (A) Schematic diagramming the U3Neo provirus in an intron. Fusion transcripts are predicted to splice to a cryptic 3′ SS located in the coding region of NPT. (B) Sequence of 3′ SS in NPT compared with the consensus sequence. Upstream exons are predicted to end in the first nucleotide of the codon to maintain the open reading frame. (C) ES cell clones containing a provirus inserted in an intron that were chosen for further analysis.

Northern blot analysis of gene trapped cell lines. Total RNA was isolated from D3 and from Ssrp1, Plk, Gas5, Lamr1, Ddx19, Mrps30, Rps19 and Plk3 mutant cell lines and hybridized to a Neo-specific probe. The D3 cell line is an uninfected ES cell line and the Ssrp1 mutant ES cell line contains a PGK-driven Neo gene. Plk and Gas5 are examples where U3NeoSV1 inserted into an exon while Lamr1, Ddx19, Mrps30, Rps19 and Plk3 all contain a provirus located in an intron. LTR: long terminal repeat.

Fusion transcripts use cryptic Neo 3′ SS in Lamr1, Ddx19, Mrps30 and Plk3 cell lines. (A) Strategy used to amplify fusion transcripts. Primers used for RT–PCR are indicated along with the U3 probe used for Southern blot analysis, which is upstream of the identified Neo 3′ SS. (B) Ethidium bromide- stained products of RT–PCR and U3 probed Southern blots. Arrows indicate spliced transcripts that use the Neo 3′ SS confirmed by sequence analysis shown on the left with the exon and Neo boundaries labeled. The sizes of the major PCR products (nucleotides) are indicated.

Use of the Neo 3′ SS leads to production of NPT fusion proteins. Whole cell protein extracts were immunoprecipitated with anti-NPT (N) specific antibody or anti-MDM (C) as a control. Immunoprecipitated proteins were subjected to SDS–PAGE and western blot analysis with anti-NPT antibody. The RSB cell line was used as a positive control and a marker for native NPT protein. Wild-type ES cells, D3, were used as a negative control. Arrows identify the NPT-specific proteins in Lamr1, Ddx19, Mrps30 and Rps19 mutant cell lines. Proteins larger than native NPT, indicative of fusion proteins, are observed in Lamr1, Ddx19 and Mrps30 mutant cell lines.

Cell–U3Neo fusion transcripts in Rps19 cells use a cryptic 3′ SS located in the intron. (A) Ethidium bromide-stained products of RT–PCR and U3 probed Southern blots. Spliced transcript hybridized to the U3 probe indicating that the Neo 3′ SS is not utilized. (B) Diagram of splice junction between U3NeoSV1 provirus (dark gray box) and the Rps19 5′ proximal exon (light gray box). The small white box indicates the single intron nucleotide (G) that is incorporated into the mature transcript. The sequence of the spliced product and the genomic sequence are shown underneath. Consensus splice sites (nearly invariant nucleotides in bold) are also given and compared with the genomic sequence, along with a putative branch point sequence. The sizes of the major PCR products (nucleotides) are indicated.

RT–PCR analysis of U3Neo fusion transcripts in Gas5 and Plk cell lines where U3NeoSV1 inserted in exons. (A) Diagram of RT–PCR strategy and possible transcripts. Neo-specific probes (NeoA and NeoB) and U3 are the same as shown in Figure , but the GSP is designed for the 5′ exon proximal to the occupied exon. (B) Ethidium bromide stain and U3 Southern blot of PCR products. Labeled arrows indicate the type of products observed referred to in (A). The sizes of the major PCR products (nucleotides) are indicated.

Generation of the U3NeoSV2 gene trap retrovirus shuttle vector. A cryptic 3′ SS located downstream of the start codon (AUG) of the Neo gene in U3NeoSV1 was removed by changing an A (shown in bold italic) to a T. This changes an invariant nucleotide in the consensus 3′ SS, shown above the U3NeoSV1 sequence. The schematic shows the structure of U3NeoSV transcripts expressed in packaging cell lines and includes a bacterial plasmid origin of replication (Ori), ampicillin resistance gene (AmpR) and BamHI and EcoRI restriction enzyme cleave sites. The neomycin resistance gene (Neo), its initiation codon (AUG) and cryptic 3′ SS in the 3′ LTR are duplicated in the integrated provirus.

Analysis of fusion transcripts expressed in cells following gene trap mutagenesis with U3NeoSV2. (A) RT–PCR strategy for amplification of cell–virus fusion transcripts. Primer NeoA was used for RT followed by amplification with NeoB and GSPs. Genomic DNA amplified with NeoB/GSP primer pair provides a PCR control and a size standard for unspliced fusion transcripts that extend from the proximal exon of the disrupted gene into the provirus. A 30 nt U3 probe was used for Southern blot analysis of PCR products. (B) PCR products amplified from fusion transcripts expressed by inserts in the Dhx9, Uhg, Tk1 and Eef1a1 genes were fractionated on 1% agarose gels and stained with ethidium bromide (RT–PCR, left) and then analyzed by Southern blot hybridization (right), probing with the U3 probe. PCR reactions were performed using total cellular RNA with (RT) and without (–RT) reverse transcription or using genomic DNA (DNA). With the exception of Uhg, the amplified fusion transcripts were smaller than the corresponding genomic DNA sequence, indicative of splicing between the upstream exon and cryptic splice sites upstream of the U3 sequence. Sequences amplified from Uhg fusion transcripts appeared to be derived mainly from transcripts extending from the proximal exon into the provirus, although some spliced products were also detected. The sizes of the major PCR products (nucleotides) are indicated.

Cryptic splice sites used for expression of fusion transcripts in Dhx9, Uhg, Tk1 and Eef1a1 mutant ES cells. RT–PCR products amplified from cell–virus fusion transcripts expressed by inserts in the Dhx9, Uhg, Tk1 and Eef1a1 genes were cloned and sequenced. RT–PCR sequences (gray boxes) are highlighted on the genomic DNA sequence, leaving intron sequences unshaded. Provirus sequences are enclosed on three sides by lines. In each case, the provirus-proximal exon of the disrupted gene (left-most gray box) spliced to a cryptic 3′ SS located completely (Dhx9 and Uhg) or partially (Tk1 and Eef1a1) in the intron sequence adjacent to the U3NeoSV2 provirus. Potential branch point sequences are underlined and gaps in the sequence (…) are indicated in nucleotides. Consensus splice site sequences are shown at the bottom for comparison.

Cell–U3His transcripts utilize a cryptic splice site in an intron of the hnRNP C gene trapped cell line. (A) Schematic of U3His insertion in the hnRNP C gene showing primers used for RT–PCR and the U1 intron-specific probe used for Southern blot analysis of PCR products. The location of the cryptic 3′ SS is also indicated. (B) Spliced transcript observed on an ethidium bromide-stained gel that is not detected with the U1 oligonucleotide probe. (C) Genomic sequence of the cryptic 3′ SS located in the hnRNP C intron compared with the consensus sequence. The putative branch point sequence is also given. Bold indicates nearly invariant nucleotides. The sizes of the major PCR products (nucleotides) are indicated.