As a first step in characterizing the molecular nature of the T^Wis allele of T, the exact identity of the ETn element inserted into the locus was determined. The primer ETn1 was designed to a region of conservation across related but different ETn. PCR was performed on yolk sac lysates from e9.5 T^Wis/T^Wis mutant embryos using the primer set F3 – ETn1 (Figure 1A). Sequence analysis of the ~1300 bp PCR product amplified from T^Wis/T^Wis DNA in two independent reactions showed that the ETn element of the T^Wis allele is identical to the 5542 bp ETn sequence, Genbank accession #Y17106, with the T^Wis ETn element inserted in reverse orientation relative to the direction of transcription of the T locus (data not shown).

The next step in characterizing the molecular nature of the T^Wis allele was to identify the aberrant transcripts produced from this allele. RT-PCR was performed on RNA from T +/+, T^Wis/+, and T^Wis/T^Wis embryos using forward and reverse primers located in different exons of the T locus, as well as a reverse primer specific to the ETn element of the T^Wis insertion (Table 1 and Figure 1B). The RT-PCR products were excised, purified, and sequenced twice on each strand with the primers used to generate them. The F2-R1 products were additionally sequenced one time each with primers F1 and R2.

The F1-R1 and F2-R1 primer pair products (F1-R1a and F2-R1a) amplified from a T +/+ embryo represented the expected wild type T RNA species (6,7,8 and 4,5,6,7,8; Figure 1B, Table 2, and Figure 3B.), while those amplified from a T^Wis/T^Wis embryo together comprised 5 aberrant transcripts (Figure 1B, Table 2, and Figure 3C.). Transcripts representing an exon 4 to exon 8 splice (4,8), an exon 5 to exon 8 splice (4,5,8), and an exon 6 to exon 8 splice (6,8) were all identified in T^Wis/T^Wis embryos. An additional mutant transcript resulted from an aberrant splicing event from exon 7 to exon 8 such that 35 nucleotides (nt) of the 3' end of exon 7 were deleted (6,7Δ35,8 and 4,5,6,7Δ35,8). Another mutant transcript included an aberrant mini-exon of 37 nt of ETn derived-sequences inserted between the 3' deleted exon 7 to exon 8 splice (6,7Δ35,+37ETn,8 and 4,5,6,7Δ35,+37ETn,8).

As expected, the F1-BGH037 primer set failed to amplify from T +/+ embryo RNA (Figure 1B). In the T^Wis/T^Wis embryo, this primer set detected a mutant transcript representing read-through transcription from exon 7 into the immediately 3' ETn insertion (6,7+Etn; Figure 1B, Table 2, Figure 3C).

In this preliminary analysis, no wild type T transcript was identified in T^Wis/T^Wis RNA using any of the primer sets. These results suggest that no T^Wis transcripts maintain normal exon 7 to exon 8 splicing, as postulated by Herrman et al. 10. To further clarify this issue, the splicing of exon 7 to exon 8 was specifically examined using the forward primer F3. This primer is located within the last 35 bp of exon 7, and therefore is deleted in all of the exon 7-containing mutant transcripts already described (see Table 1, Table 2, and Figure 3). RT-PCR analysis with this primer confirmed that while the F1-BGH037 transcript is only produced off the T^Wis allele, the F3-R1 transcript is only produced off of the wild type T allele (Figure 2).

In some cases, a prominent band larger than the expected wild type size was amplified by the F3-R1 primers from T^Wis/T^Wis embryo RNA (Figure 2). Sequence analysis showed that this larger T^Wis/T^Wis specific band represented two additional aberrant transcripts (Table 2 and Figure 3C). Notably, these two products were never observed in T^Wis/+ RNA (Figure 2). A shorter variant contained the intact 3' end of exon 7 and 60 nt of read-through transcription into the ETn element spliced to exon 8 (7+60ETn,8). Given that the BGH037 primer is contained within the 60 nt of ETn read-through, this species is not necessarily distinct from the 6,7+ETn transcript amplified by F1-BGH037, as both could be amplified from the same hypothetical RNA 6,7+60ETn,8. The longer variant contained the intact 3' end of exon 7 and 60 nt of read-through into the ETn element spliced to a 37 nt mini-exon of ETn sequence, then spliced to exon 8 (7+60ETn,+37ETn,8). The 37 nt ETn mini-exon was the same sequence found in the 4,5,6,7Δ35,+37ETn,8 and 6,7Δ35,+37ETn,8 products described above. This species is also not necessarily distinct from the 6,7+ETn transcript amplified by F1-BGH037, as both could be amplified from the same hypothetical RNA 6,7+60ETn,+37ETn,8. Alternatively, the 6,7+ETn product might have amplified from an RNA 6,7+?ETn, where an unknown amount of the ETn insertion was transcribed and the transcript terminated in ETn sequence without being spliced to exon 8. Overall, wild type exon 7 to exon 8 splicing was never identified in transcripts produced from the T^Wis allele of T.

The characterization of T^Wis allele transcripts presented here confirms that the intron 7 donor splice site is indeed non-functional such that wild type T mRNA is never produced. Furthermore, the exon 1–7+ETn transcript predicted by Herrmann et al. 10 was identified. However, at least 7 other T^Wis mutant transcripts are also produced (Figure 3C). All of these transcripts serve to bypass the mutated splice donor site; either by splicing over exon 7, activating a cryptic splice site within exon 7, or transcribing through exon 7 into the adjacent ETn sequences. The cryptic exon 7 splice site used,

CCT

C/A,AG

Genetically, the T^Wis allele acts as a dominant negative in that T^Wis /T^Wis embryos have a more severe phenotype than the null T/T embryos. However, the biochemical nature of this dominant negative effect is not known. This study confirms previous work 13 showing the existence of a transcript that would result in a truncated protein if translated. Because the DNA-binding T-domain would be intact, this hypothetical protein could compete with the wild type protein producing a dominant negative effect. Alternatively, the presence of heterologous Etn element sequences in T^Wis transcripts may stimulate posttranscriptional gene silencing by an RNA-induced silencing complex (RISC)-based RNAi degradation pathway which could target degradation of wild type transcripts 14.

The T^Wis allele was maintained on a mixed A/J + C57BL/6 + 129/SvEv background. Embryos were collected from timed heterozygous matings. At e7.5 and e8.5, a portion of the extraembryonic region was removed for PCR genotyping, and the remainder of the embryos placed individually into 400 μL TRIZOL Reagent (Life Technologies, Inc.) for RNA preparation. At e9.5, mutant embryos were identified visually by absence of posterior embryonic tissues 13. For genomic PCR of the ETn insertion, yolk sac lysates from homozygous mutant e9.5 embryos were used. Genomic DNA from a wild type mouse served as a negative control. PCR was performed in 50 μL reactions containing: template, 50 pmol each of primers F3 (5'-CATAACGCCAGCCCACCTACTG-3') and ETn1 (5'-CACGATTTGTGGGTAAAATAGGAG C-3'), 2.5 units Taq DNA polymerase, 0.2 mM dNTPs, 20 mM TrisHCl (pH 8.4), 50 mM KCl, and 1.5 mM MgCl. Cycling parameters were as follows: initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 67°C for 45 sec, and extension at 72°C for 90 sec, and a final extension at 75°C for 5 min. PCR products were electrophoresed through a 1.3% agarose TAE gel, excised from the gel, and purified using QIAEX II Gel Extraction Kit (Qiagen). Purified products were sequenced twice on each strand with the F3 and ETn1 primers using an ABI Prism Model 373 Automated DNA Sequencer. The ETn elements used to design the Etn1 primer were Genbank #s Y17106, Y17107, and X15598.

For genotyping of e7.5 and e8.5 embryos, PCR was performed on lysates of extraembryonic tissue as template, using primers BGH037 (5'-ACGTTGCGAGCTGCTGCGGC-3'), BGH039 (5'-ACCCATGTCAAACCCATCAG-3'), and BGH052 (5'-CCTATGCGGACAATTCATCTG-3'), in a similar reaction mix except for 1.0–1.5 mM MgCl. Cycling parameters were as follows: initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 61° for 45 sec, and extension at 72°C for 45 sec, and a final extension at 75°C for 3 min. The primers BGH052 and BGH037 amplify an ~180 base pair (bp) fragment from the T^Wis insertion allele, while primers BGH052 and BGH039 amplify an ~220 bp fragment from the wild type allele.

For RT-PCR, total RNA from individual e7.5 and e8.5 embryos was prepared essentially according to the TRIZOL Reagent manufacturer's protocols for isolation of RNA from small quantities of tissue. Total RNA was reverse transcribed using Superscript II RNase H^- Reverse Transcriptase (Life Technologies, Inc.) as per manufacturer's protocol, except that all reactions were doubled to 40 μL final volume. RT reactions were performed on one half or one third of the total RNA isolated from an e7.5 or e8.5 embryo, respectively. For positive control reactions, RNA from one wild type embryo was used. Standard RT-PCR reactions were performed with each 50 μL reaction containing 1/8 of an RT reaction, 50 pmol each of forward and reverse primer, 2.5 units Taq DNA polymerase, 0.2 mM dNTPs, 20 mM TrisHCl (pH 8.4), 50 mM KCl, and 1.5 mM MgCl. Cycling parameters were as follows: initial denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 30 sec, annealing at 65° or 64°C for 45 sec, and extension at 72°C for 45 sec, and a final extension at 75°C for 3 min. RT-PCR products were excised from gels, purified using the QIAEX II Gel Extraction Kit (Qiagen), and sequenced in an ABI Prism Model 373 Automated DNA Sequencer. Primers used for RT-PCR and/or sequencing were: F1 (5'-GTCATCGCCCTACCCCAG-3'), R1 (5'-GTGTGCGTCAGTGGTGTGTAATG-3'), F2 (5'-CCCATTTGCTAAAGCCTTCCTTG-3'), R2 (5'-AGGCACTCCGAGGCTAGACCAG-3'), BGH037 (5'-ACGTTGCGAGCTGCTGCGGC-3'), and F3 (5'-CATAACGCCAGCCCACCTACTG-3'). RT-PCR reactions using primers Hprt1a (5'-CCTGCTGGATTACATATAAGCACTG-3') and Hprt1b (5'-GTCAAGGGCATATCCAACAACAAAC-3') served as positive controls amplifying a 354 bp product from the ubiquitously expressed hprt transcript 15.