INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

All cells require protein synthesis followed by transport and correct targeting of these proteins to their proper destinations. Biochemical, genetic, and intracellular localization studies of kinesin motors have suggested that some of these motor proteins may power intracellular transport events in neurons (1, 6, 7). As microtubule-dependent motors, members of the kinesin superfamily share extensive sequence similarity within the motor domain but display diversification in their tail domains. The motor domain is composed of a catalytic domain that hydrolyzes ATP and interacts with the microtubule track and a short neck domain important for processive movement and control of direction. The tail domains have been suggested to provide different cargo-binding or regulatory partners and to confer the ability to form different types of oligomers (21).

The kinesin II holoenzyme was first identified in sea urchins and subsequently identified in most species and found to be composed of two different motor subunits from the Kif3 family (3). Genetic and localization experiments in Chlamydomonas, Tetrahymena thermophila (4, 22), Caenorhabditis elegans (19), sea urchins (5), and mice (9, 23, 24) suggest that kinesin II in many cases is essential for the construction and maintenance of motile and nonmotile cilia and flagella (11, 18). In Chlamydomonas, kinesin II appears to transport a large protein complex, termed a raft, possibly with protein cargoes attached, from the sites of synthesis in the cell body to the sites of utilization at the tip of the flagellum (4, 10, 16).

Three members (Kif3A, Kif3B, and Kif3C) of the Kif3 family have been characterized in mice (9, 23, 24). Kif3A was previously reported to form a heterodimer with Kif3B or Kif3C, but Kif3B and Kif3C cannot associate with each other (14, 24). Mouse mutants lacking either the Kif3A or Kif3B gene resulted in embryonic lethality and embryonic ciliary morphogenesis defects (13, 15, 20), suggesting that they also play roles in ciliary morphogenesis in mammals. When Kif3A was specifically deleted from retinal photoreceptors using the Cre-loxP system, complete loss of Kif3A caused large accumulations of opsin, arrestin, and membranes within the photoreceptor inner segment, which suggests that kinesin II is required to transport opsin and arrestin from the inner to the outer segments (12). In contrast to Kif3A and Kif3B, whose expression is ubiquitous, Kif3C expression is highly enriched in both the central and peripheral nervous systems. Intracellular localization and biochemical studies suggest that Kif3C is an anterograde motor which may be involved in anterograde axonal transport (14, 24). Nevertheless, the precise in vivo functions of Kif3C remain unknown.

To understand the in vivo function of the Kif3C gene, we used homologous recombination in embryonic stem cells to construct two types (Kif3C^typeI and Kif3C^null) of knockout mouse strain for the Kif3C gene. In the Kif3C^typeI mice, the motor region and half of the -helical coiled-coil domain of the Kif3C protein were removed, but the rest of the -helical coiled-coil domain and the tail of the protein still remained. In the Kif3C^null mice, no Kif3C mRNA or protein can be detected by Northern and Western blotting, which suggests that homozygous Kif3C^null mice are null mutants. Both homozygous Kif3C^typeI and Kif3C^null mutants are viable, reproduce normally, and apparently develop normally. These results suggest that Kif3C is dispensable for normal neural development and behavior in the mouse.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Cloning and mapping of the Kif3C gene. A 1.5-kb Kif3C cDNA fragment encoding the motor domain of the Kif3C protein was used for isolating Kif3C genomic clones from a mouse 129/SV/J genomic phage library (a gift from the lab of Jamey Marth). The genomic clones we isolated from the library were then cloned into the vector Bluescript (Stratagene, La Jolla, Calif.). The map of the Kif3C gene was obtained by digestion of the genomic clones using different restriction enzymes and probing with different regions of the Kif3C cDNA. Southern blotting and other molecular biological techniques were performed according to standard methods.

Generation of targeting vectors and ES cells. We made two targeting vectors. One vector was built using pflox (a gift from the lab of Jamey Marth) (2). In this vector we deleted a 2.1-kb DNA fragment between BamHI and EcoRV of the Kif3C gene (Fig. 1A). The DNA fragment contains the first exon (2.0 kb) and part of the first intron (0.1 kb) of the Kif3C gene and encodes amino acid residues 1 to 518 of the Kif3C protein. The linearized targeting construct with NdeI was introduced into R1 embryonic stem (ES) cells via electroporation (8) prior to selection with 250 µg of active G418/ml for 7 to 9 days. ES clones resistant to G418 were analyzed by Southern analysis. When the ES cell genomic DNA was digested with BamHI and probed with a 600-bp DNA fragment between two NdeI cut sites (Fig. 1A), the wild-type and targeted Kif3C alleles (type III) were detected as 9- and 26-kb bands, respectively (Fig. 1B). The targeted Kif3C allele was confirmed by Southern blotting with different restriction enzymes and probes. ES cell clones bearing a targeted Kif3C allele (type III) were electroporated with Cre expression plasmid pCre-Hygro (a gift from the lab of Jamey Marth) to excise the DNA fragment flanked by loxP sites (2). ES clones resistant to ganciclovir (2 µM) were analyzed by Southern analysis in which the genomic DNA was digested with EcoRI and probed with a loxP fragment (a gift from the lab of Jamey Marth). Two types of ES cells were obtained (Fig. 1A and C). One, bearing only one copy of the loxP sequence, is called Kif3C^typeI, in which the 2.1-kb Kif3C DNA fragment was deleted. The other, containing two copies of the loxP sequence, is called Kif3C^typeII, in which the Kif3C gene should function normally because the two loxP fragments were inserted in an intron and the 5' nontranslated region and did not alter other aspects of the Kif3C gene.

View larger version (36K): [in this window] [in a new window]   FIG. 1.   Generation and analysis of Kif3CtypeI and Kif3CtypeII mutants in ES cells. (A) Strategy for generating Kif3CtypeI and Kif3CtypeII mutants. A 2.1-kb DNA fragment between BamHI and EcoRV of the Kif3C gene was flanked with two loxP fragments. A PGK-neo and PGK-tk selection marker with a third loxP fragment was added into the vector. (B) Southern analysis of targeted ES cells (Kif3CtypeIII). ES cell genomic DNA was cut with BamHI and probed with a 600-bp NdeI DNA fragment (probe A). The wild-type and targeted Kif3CtypeIII alleles were detected as 9- and 26-kb bands, respectively. (C) Southern analysis of Cre-transfected ES cells. Kif3CtypeIII (+/) ES cells were transfected with the Cre plasmid to excise the DNA fragment flanked by loxP sites. ES clones resistant to ganciclovir were analyzed by Southern analysis, in which the genomic DNA was digested with EcoRI and probed with a loxP fragment (probe B). Each band represents a loxP fragment. Two types (Kif3CtypeI and Kif3CtypeII) of ES cells were obtained.

Generation of Kif3C targeted mice. Three types (Kif3C^typeI, Kif3C^typeII, and Kif3C^null) of ES cells were used for generating chimeric mice (129/SvJ-derived ES cells in blastocysts of C57BL/6J mice) as previously described (8). When the chimeras were backcrossed to C57BL/6J mice, heterozygous Kif3C^typeI (+/), Kif3C^typeII (+/), and Kif3C^null (+/) mice were obtained. These heterozygous mice were used for interbreeding to produce homozygous Kif3C^typeI (/), Kif3C^typeII (/), and Kif3C^null (/) mice, respectively. The genotypes of animals were obtained by Southern blotting or PCR as indicated.

Analysis of Kif3C targeted mice. Gross and histopathological analyses employed standard techniques. Littermate Kif3C (+/+) and either Kif3C^typeI (/) or Kif3C^null (/) mice were examined for appearance, posture, circadian activity, home cage assessment, rotating rod task performance, and balance. These behavioral tests were carried out using standard protocols.

Western analysis and immunoprecipitation. Monoclonal anti-Kif3A and polyclonal anti-Kif3B antibodies were from Babco. Monoclonal anti-actin antibody was from Boeihringer Mannheim. Monoclonal antibody SUK4 is directed against the kinesin heavy chain. Affinity-purified rabbit antibodies against the C terminus of mouse Kif3C were prepared and Western analysis and immunoprecipitation of mouse brain lysates were performed as described previously (24).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Generation and analysis of Kif3C^typeI and Kif3C^typeII mice. To explore the in vivo function of the Kif3C gene, we first made a targeting vector to delete a 2.1-kb DNA fragment of the Kif3C gene in ES cells. Because we were concerned that Kif3C^typeI (/) mice might be inviable, Kif3C^typeII (/) mice, which are conditional (tissue specific) knockout mice, would serve for functional analysis of the Kif3C gene. As described in Materials and Methods, we obtained both Kif3C^typeI (+/) and Kif3C^typeII (+/) ES cells (Fig. 1A). When both Kif3C^typeI (+/) and Kif3C^typeII (+/) ES cells were injected into the blastocysts of C57BL/6J mice, several chimeras were generated. These chimeras were successfully used to generate homozygous Kif3C^typeI (/) and Kif3C^typeII (/) mice, respectively. Both homozygous Kif3C^typeI (/) and Kif3C^typeII (/) mice were normal and healthy, just like their wild-type littermates. The ratios of wild-type Kif3C (+/+), heterozygous Kif3C^typeI (+/) or Kif3C^typeII (+/), and homozygous Kif3C^typeI (/) or Kif3C^typeII (/) mice from the heterozygous parents yielded the predicted Mendelian ratios of 1:2:1 expected for nonlethal alleles. Thus, the Kif3C^typeI (/) and Kif3C^typeII (/) pups were no less viable than their wild-type and heterozygous littermates.

View larger version (31K): [in this window] [in a new window]   FIG. 2.   Analysis of Kif3CtypeI and Kif3CtypeII mice. (A) Western analysis of Kif3CtypeII mice. Brain lysates from Kif3CtypeII (/), Kif3CtypeII (+/), and wild-type (+/+) littermates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The duplicate blots were probed with antibodies against Kif3A, Kif3B, and Kif3C. (B) Northern analysis of Kif3CtypeI mice. Total RNA was isolated from the Kif3CtypeI mouse brains and analyzed in a formaldehyde-agarose gel. The duplicate blots were probed with cDNA of Kif3A, Kif3B, and Kif3C. Two different probes, encoding either the motor region or the tail region as indicated, were used for Kif3C. (C) Western analysis of Kif3CtypeI mice. Brain lysates from Kif3CtypeI mice were analyzed by SDS-PAGE. The duplicate blots were probed with antibodies against Kif3A, Kif3B, and Kif3C and a monoclonal antibody, SUK4, for kinesin heavy chain. The antibodies against Kif3C detected a truncated Kif3C protein. (D) Immunoprecipitation-Western analysis of Kif3CtypeI mice. Brain lysates from Kif3CtypeI mice were immunoprecipitated with anti-Kif3C or anti-Kif3B antibodies or immunoglobulin G (IgG) as a control. The immunoprecipitated samples were analyzed by SDS-PAGE. The same blot was probed and reprobed with antibodies against Kif3A, Kif3B, and Kif3C. The samples in the left three lanes were from Kif3CtypeI (/) mice and the samples in the right three lanes were from wild-type littermates. (The truncated Kif3C protein in wild-type mice was probably generated from protein degradation.)

Generation and analysis of Kif3C^null mice. Using standard methods, we used Kif3C^null (+/) ES cells (Fig. 3A) to generate Kif3C^null (/) and wild-type littermates. In the Kif3C^null (/) mice, a 4.5-kb DNA fragment of the Kif3C gene was deleted and replaced by a drug resistance cassette and a poly (A) signal. The 4.5-kb DNA fragment encodes amino acid residues 1 to 518 of the Kif3C protein. This deletion includes the whole motor region and half of the -helical coiled-coil domains of the motor domain similar to the Kif3C^typeI (/) mice. The deletion was confirmed by PCR (Fig. 4A) and by Western blotting (Fig. 4B). In genomic PCR of the Kif3C mice, a 500-bp DNA fragment which represents the Kif3C wild-type allele was found for both the wild-type (+/+) and heterozygous (+/) mice but not for the homozygous mutant Kif3C^null (/) mice (Fig. 4A). In contrast, a 400-bp DNA fragment amplified from PGK-neo which represents the Kif3C mutated allele was found for both the heterozygous Kif3C^null (+/) and homozygous Kif3C^null (/) mutants but not for the wild type (+/+) mice (Fig. 4A). The results from PCR analysis were confirmed by Southern analysis using the same enzyme and probe as described for Fig. 3B (data not shown). In Western analysis of the brain lysates from the Kif3C^null (/) mice and wild-type littermates, no Kif3C-specific protein was detected in the homozygous mutants using antibodies against the C terminus of the Kif3C protein. These results clearly indicate that the mice we generated are truly null for the Kif3C gene.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Generation and analysis of Kif3Cnull mutants in ES cells. (A) Strategy for generating Kif3Cnull mutants. One 4.5-kb DNA fragment between BamHI and XhoI of the Kif3C gene was replaced with a 1.7-kb PGK-neo cassette. The targeting vector included a negative selection marker, PGK-tk. The targeting vector was linearized with XhoI and introduced into R1 ES cells. (B) Southern analysis of ES cells. ES cell genomic DNA was cut with XbaI and probed with a 1.5-kb BamHI/EcoRI DNA fragment. The wild-type (WT) and targeted Kif3C (Mut) alleles were detected as 10.0- and 7.5-kb bands, respectively. P1, P2, P3, and P4 indicate the locations of primers used for genotyping Kif3Cnull mice (Fig. 4).

View larger version (26K): [in this window] [in a new window]   FIG. 4.   Analysis of Kif3Cnull knockout mice. (A) PCR analysis of the Kif3Cnull knockout mice. A set of four primers was used for genotyping the Kif3Cnull mice. Two primers based on Kif3C sequences (forward P1, GGT CAT GAG CAG ATT CTG AC; reverse P2, GAG AGC TGA CCT CAT TCA TG) were used for PCR to amplify a 500-bp DNA fragment which is absent from the Kif3Cnull (/) mutant locus. Another two primers based on the neo sequence (forward P3, GAT GGA TTG CAC GCA GGT TCT; reverse P4, AGG TAG CCG GAT CAA GCG TAT) were used for amplifying a 400-bp DNA fragment which is missing in the wild-type Kif3C locus. The positions of PCR primers are indicated in Fig. 3A. (B) Western analysis of Kif3Cnull knockout mice. Brain lysates from the Kif3Cnull mice were analyzed by SDS-PAGE. The blot was probed with antibodies against Kif3C, which was made using the C terminus of the Kif3C protein. No truncated Kif3C protein was detected. WT, wild type; Mut, targeted Kif3C alleles. Numbers to the right of panel B indicate molecular mass, in kiloDaltons.

View larger version (72K): [in this window] [in a new window]   FIG. 5.   Morphology of brain from wild-type (A) and Kif3Cnull (/) (B) mice. Brains were isolated from adult animals perfused with paraformaldehyde, postfixed, and embedded in paraffin. Sections were stained with cresyl violet.

Functions of Kif3C motor. The neural-specific expression of Kif3C had suggested that Kif3C would play an important role in the nervous system. However, when we disrupted the Kif3C gene in mice, the nervous system apparently functioned normally in the homozygous mutants. This conclusion is supported by behavioral tests and histological analyses in which we found no difference between the Kif3C knockout mice and their wild-type littermates.

View larger version (41K): [in this window] [in a new window]   FIG. 6.   Western analysis of Kif3Cnull (/) mice. Brain lysates from Kif3Cnull mice were analyzed by SDS-PAGE. The duplicate blots were probed with antibodies against Kif3A (A), Kif3B (B), Kif3C (C), and actin (D).