Molecular cloning and functional analysis of mouse C-terminal kinesin motor KifC3

Abstract

Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. To study the molecular mechanism of intracellular transport involving microtubule-dependent motors, a cDNA encoding a new kinesin-like protein called KifC3 was cloned from a mouse brain cDNA library. Sequence and secondary structure analysis revealed that KifC3 is a member of the C-terminal motor family. In contrast to other mouse C-terminal motors, KifC3 is apparently ubiquitous and may have a general role in intracellular transport. To understand the in vivo function of the KifC3 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC3 gene. Homozygous mutants of the KifC3 gene are viable, reproduce normally, and apparently develop normally. These results suggest that KifC3 is dispensable for normal development and reproduction in the mouse.

FIG. 1

Sequence analysis of KifC3. Predicted amino acid sequence alignment of KifC3 with KifC1, KifC2, hKifC3, and fKif2. Identical amino acids and conserved, but not identical, amino acids in the five polypeptides are shown in black and shaded boxes, respectively. The alignment was performed with the UWGCG software package (4) and boxed with the program BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html). These sequence data are available from EMBL and GenBank under the following accession numbers: KifC1, D49544; KifC2, U92949; KifC3, AF013118; hKifC3, AF004426; and fKifC2, U64819.

FIG. 2

Analysis of expression of KifC3 in mouse tissues and cell lines. A set of duplicate Northern blots was probed with KifC3 (A) (two transcripts of 3.3 and 2.3 kb were detected in most mouse tissues and cell lines), KifC4 (B) (one transcript of 2.3 kb was detected only in ES cells and testis), and KifC3 (C) (two transcripts of 7.2 and 4.3 kb were detected only in brain).

FIG. 3

Generation and analysis of KifC3 mutants in ES cells. (A) Strategy for generating KifC3 knockout mice. One 6.5-kb DNA fragment between ClaI and BglII was replaced with an SA-IRES-βgeo cassette. This cassette contains an en-2 SA, an IRES, and a fusion of lacZ and neo genes (βgeo). The targeting vector was linearized with SalI. (B) Southern analysis of ES cells. The wild-type (WT) and targeted KifC3 (Mut) alleles were detected as 10- and 8-kb bands, respectively, in Southern blot analysis of ES cell genomic DNA cut with EcoRI (RI) and probed with a 1.0-kb SalI/RcoRV fragment. RV, RcoRV; Bam, BamHI.

FIG. 4

Analysis of KifC3 knockout mice. (A) PCR and Southern analysis of the KifC3 knockout mice. The primers for PCR and probe for Southern analysis are described in Materials and Methods. (B) Northern analysis of KifC3 knockout mice. Total RNA was isolated from the kidney and testis of KifC3 mice (+/+, +/−, and −/−), and the Northern blot was probed with a cDNA fragment, which corresponds to the deleted region of the KifC3 gene.

FIG. 5

Morphology of kidney tissue from wild-type (A, C, and E) and knockout (B, D, and F) mice. Kidneys from adult animals were isolated, fixed in paraformaldehyde, and embedded in paraffin. Sections were stained with hematoxylin and eosin. Bar = 100 μm (A to D) and 50 μm (E and F).