FIG. 2.
The deleted knt allele is a null allele. (A) RT-PCR analysis of knt RNA obtained from kntΔ/Δ and knt+/+ EF. PCR with primers located in exon 2 and exon 6 (ex2-ex6, left) yields amplification products of 273 bp (kntΔ/Δ) and 716 bp (knt+/+), indicative of splicing of exon 2 directly to exon 6 at the kntΔ/Δ mRNA. This results in a frameshift mutation and generates a PTC. That exon 2 to exon 6 join in the kntΔ/Δ transcripts was verified by DNA sequencing. PCR encompassing exons 4 to 6 (ex4-ex6, middle) yields a 400-bp product in the wild type. No amplification is obtained on the kntΔ/Δ template due to the engineered deletion of exons 3 to 5. Splicing of exon 6 to exon 7 occurs in transcripts from the deleted and the wild-type knt alleles (ex6-ex7, right), yielding products of 165 bp. For primer sequences, see Material and Methods. Lanes 1, with RT; lanes 2, without RT; lanes 0, negative controls (no template). (B) Northern blot analysis, verifying the absence of stable knt transcripts in kntΔ/Δ mice. The same RNA preparations as used in panel A were hybridized to knt5′ and knt3′, two cDNA probes encompassing the entire coding sequence of the knt transcript. The blots were stripped and rehybridized to glctrp (mouse facilitated glucose transporter) as a loading control. kntΔ/Δ transcripts are rapidly degraded, most probably due to nonsense mediated decay. (C) Western blot analysis showing the absence of KNT protein form kntΔ/Δ mice. Organ homogenates of kidney, spleen, and brain tissues obtained from mice of the indicated genotypes were probed with a polyclonal anti-KNT serum (RS-TUM) directed against the central part of the KNT protein. Black arrow, 160-kDa full-length KNT; open arrow, 120-kDa form of KNT (27, 32); open triangle, irrelevant protein. (D) Immunolabeling of EF with RS-TUM shows the absence of KNT from kntΔ/Δ EF. Magnification, ×400.