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. 2001 Mar;21(5):1444-52.
doi: 10.1128/MCB.21.5.1444-1452.2001.

Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development

L M Esteban  1 , C Vicario-Abejón, P Fernández-Salguero, A Fernández-Medarde, N Swaminathan, K Yienger, E Lopez, M Malumbres, R McKay, J M Ward, A Pellicer, E Santos
Affiliations

Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development

L M Esteban et al. Mol Cell Biol. 2001 Mar.

Abstract

Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras(+/-) mice produced H-ras(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-ras did not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras(-/-) mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras(-/-) mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras(-/-)/N-ras(-/-)) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-than-expected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development.

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Figures

FIG. 1
FIG. 1
Targeted disruption of the murine H-ras gene in ES cells and mice. (A) Schematic representation of the H-ras locus and targeting vector. Boxes in the wild-type allele schematics represent the exons of the H-ras gene. The open boxes in the targeting vector schematics represent the pgk-neo and pgk-tk selectable marker genes. The position of the 3′ flanking probe used in Southern blotting is indicated. (B) Homologous recombination of the targeting vector in mice was verified by Southern blotting, digesting genomic DNA with BglII, and hybridizing with a 3′-flanking probe. The wild-type allele produced a 10.5-kb band, whereas the mutant allele yielded a 6.5-kb band due to the introduction of a new BglII site in the targeting vector. (C) Routine genotyping of mice was performed by PCR using the oligonucleotides indicated, whose sequences are given in Materials and Methods. The LM88 and LM89 primers are specific for the H-ras gene and amplified a 434-bp fragment. The LM82 primer is specific for the Neo-PGK promoter and amplified a 336-bp fragment with LM88. Pv, PvuII; H, HindIII; Bg, BglII.
FIG. 2
FIG. 2
Detection by RT-PCR of H-ras, N-ras, K-ras4A, and K-ras4B in total RNA from tissues of animals with wild-type and mutant H-ras. Oligonucleotides specific for detecting each of the different ras gene transcripts were used as indicated. Their sequences are detailed in Materials and Methods. (Above) LM99 and LM111 amplified H-ras, and LM164 and LM165 amplified N-ras. (Below) LM209 and LM210 amplified K-ras4A, and LM209 and LM211 were specific for K-ras4B. Tissues used were brain (B), liver (L), kidney (K), and testis (T) from mice with wild-type (+/+) and null (−/−) H-ras genes. The levels of expression of N-ras and both K-ras4A and K-ras4B molecules in tissues were not affected by the absence or presence of H-ras gene products. As previously described, K-ras4A shows different expression levels depending on the tissues studied. The oligonucleotides used for K-ras4B occasionally lighted up an alternative band in liver and kidney whose intensity was not affected by either the presence or the absence of H-ras.
FIG. 3
FIG. 3
Neuronal differentiation is not altered in neurons lacking H-ras. Cultured hippocampal neurons from wild-type mice (A, C, E, G, I, K) and H-ras−/− mice (B, D, F, H, J, L) were stained with specific antibodies against MAP-2ab, GABA, calretinin, synapsin I, phosphorylated CaMKIIα (PCaMKIIα), or CaMKIIα. Scale bar: A, B, and G to J, 35 μm; C to F, 55 μm; K and L, 25 μm.
FIG. 4
FIG. 4
Analysis of double mutant mice deficient for H-ras and/or N-ras. (A) Genotyping of representative animals resulting from crossing of H-ras- and N-ras-disrupted mice. Oligonucleotides used were as described in Materials and Methods: LM82, LM88, and LM89 were used for the H-ras gene, and LM164, LM205, and LM166 were used for the N-ras gene. Note that mouse A97 was deficient in both gene loci. (B) RT-PCR detection of expression of the different ras genes. Procedures were exactly as those used for Fig. 2. For K-ras, only K-ras4B expression is shown since the levels of K-ras4A were rather low in the brain and were similar for all four genotypes studied here (data not shown). (C) Western immunoblotting of H-Ras, N-Ras, and K-Ras proteins in double and single mutation mice. A 50-μg sample of total protein from brain was electrophoresed on SDS–14% polyacrylamide gels. The antibodies used were polyclonal anti-H-Ras and anti-N-Ras antibodies and monoclonal anti-K-Ras antibodies (recognizing both K-Ras4A and K-Ras4B proteins). (B and C) Representative examples showing only expression of RNA and protein from brain. Other tissues analyzed yielded similar results, with no increase in the expression of K-Ras in the double mutation mice compared with mice with single mutations of H-Ras or N-Ras or with wild-type mice.
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References

    1. Apolloni A, Prior I A, Lindsay M, Parton R G, Hancock J F. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol Cell Biol. 2000;20:2475–2487. - PMC - PubMed
    1. Bonni A, Brunet A, West A E, Datta S R, Takasu M A, Greenberg M E. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science. 1999;286:1358–1362. - PubMed
    1. Bos J L. ras oncogenes in human cancer: a review. Cancer Res. 1989;49:4682–4689. - PubMed
    1. Brambilla R, Gnesutta N, Minichiello L, White G, Roylance A J, Herron C E, Ramsey M, Wolfer D P, Cestari V, Rossi-Arnaud C, Grant S G, Chapman P F, Lipp H P, Sturani E, Klein R. A role for the Ras signalling pathway in synaptic transmission and long-term memory. Nature. 1997;390:281–286. - PubMed
    1. Brown K, Bailleul B, Ramsden M, Fee F, Krumlauf R, Balmain A. Isolation and characterization of the 5′ flanking region of the mouse c-Harvey-ras gene. Mol Carcinog. 1988;1:161–170. - PubMed

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