K-ras is an essential gene in the mouse with partial functional overlap with N-ras

Abstract

Mammalian ras genes are thought to be critical in the regulation of cellular proliferation and differentiation and are mutated in approximately 30% of all human tumors. However, N-ras and H-ras are nonessential for mouse development. To characterize the normal role of K-ras in growth and development, we have mutated it by gene targeting in the mouse. On an inbred genetic background, embryos homozygous for this mutation die between 12 and 14 days of gestation, with fetal liver defects and evidence of anemia. Thus, K-ras is the only member of the ras gene family essential for mouse embryogenesis. We have also investigated the effect of multiple mutations within the ras gene family. Most animals lacking N-ras function and heterozygous for the K-ras mutation exhibit abnormal hematopoietic development and die between days 10 and 12 of embryogenesis. Thus, partial functional overlap appears to occur within the ras gene family, but K-ras provides a unique and essential function.

Figure 1

Disruption of K-ras in ES cells. (A) The K-ras targeting vector pK-ras KO was constructed by inserting fragments from intron 0 and intron 1 of the mouse K-ras gene into the plasmid pPNT. The regions of homology consist of a 2.8-kb NotI–SalI fragment and a 5.1-kb HindIII–KpnI fragment. Both the pkg-neo and HSV-tk cassettes were positioned such that they were transcribed in the same transcriptional orientation as K-ras. (B) Southern blot analysis of BamHI plus StuI-digested genomic DNA from ES cell clones using a probe 3′ to the region of homology (3′ ext probe). Lanes 3–6 represent four independent K-ras^+/− ES cells clones, as they possess both an 8.1-kb wild-type (wt) allele and a 7.0-kb mutant-specific K-ras allele. (Lane 1) The DNA is from wild-type ES cells; (lane 2) from a nonhomologous integrant; and (lanes 7,8) two independent K-ras^−/− mutant ES cell clones that were obtained after exposure to increasing concentrations of G418. (C) Southern blot analysis of StuI-digested genomic DNA using a probe specific for neo. On a parallel set of samples to C the neo probe detected the expected 8.4-kb StuI fragment, as well as an additional 6.9-kb fragment in all K-ras^+/− ES cell clones (lanes 3–6). As expected, no signal was detected in the wild-type ES cells (lane 1), and the lower band depicted in lane 2 represents the random integration pattern for this nonhomologous integrant. A number of different digests were performed and screened with the above probes, as well as with probes 5′ to the region of homology, within the 5′ region of homology, and spanning the PGK promoter sequence to determine the actual configuration of the mutant allele. Both the expected and actual K-ras mutant allele configurations are shown in A. This was confirmed further by PCR analysis using the primer pairs shown in A. Primer pairs 3′ neo + 5′ neo and 3′ homolog + 5′ homolog specifically amplify a 5.1- and a 1.8-kb fragment, respectively, as would be expected for this configuration. This head-to-tail integration pattern occurred either one time (lanes 3,5) or multiple times (B lanes 4,6). (D) PCR analysis of E12.5 embryos derived from a K-ras^+/− intercross showing the presence of K-ras^−/− embryos (lanes marked with an asterisk). The two alleles can be distinguished using primer pairs that specifically amplify a 360-bp wild-type fragment (5′ I0 + 3′ Ex1) or a 270-bp mutant specific fragment (5′ I0 + 3′ neo).

Figure 1

Disruption of K-ras in ES cells. (A) The K-ras targeting vector pK-ras KO was constructed by inserting fragments from intron 0 and intron 1 of the mouse K-ras gene into the plasmid pPNT. The regions of homology consist of a 2.8-kb NotI–SalI fragment and a 5.1-kb HindIII–KpnI fragment. Both the pkg-neo and HSV-tk cassettes were positioned such that they were transcribed in the same transcriptional orientation as K-ras. (B) Southern blot analysis of BamHI plus StuI-digested genomic DNA from ES cell clones using a probe 3′ to the region of homology (3′ ext probe). Lanes 3–6 represent four independent K-ras^+/− ES cells clones, as they possess both an 8.1-kb wild-type (wt) allele and a 7.0-kb mutant-specific K-ras allele. (Lane 1) The DNA is from wild-type ES cells; (lane 2) from a nonhomologous integrant; and (lanes 7,8) two independent K-ras^−/− mutant ES cell clones that were obtained after exposure to increasing concentrations of G418. (C) Southern blot analysis of StuI-digested genomic DNA using a probe specific for neo. On a parallel set of samples to C the neo probe detected the expected 8.4-kb StuI fragment, as well as an additional 6.9-kb fragment in all K-ras^+/− ES cell clones (lanes 3–6). As expected, no signal was detected in the wild-type ES cells (lane 1), and the lower band depicted in lane 2 represents the random integration pattern for this nonhomologous integrant. A number of different digests were performed and screened with the above probes, as well as with probes 5′ to the region of homology, within the 5′ region of homology, and spanning the PGK promoter sequence to determine the actual configuration of the mutant allele. Both the expected and actual K-ras mutant allele configurations are shown in A. This was confirmed further by PCR analysis using the primer pairs shown in A. Primer pairs 3′ neo + 5′ neo and 3′ homolog + 5′ homolog specifically amplify a 5.1- and a 1.8-kb fragment, respectively, as would be expected for this configuration. This head-to-tail integration pattern occurred either one time (lanes 3,5) or multiple times (B lanes 4,6). (D) PCR analysis of E12.5 embryos derived from a K-ras^+/− intercross showing the presence of K-ras^−/− embryos (lanes marked with an asterisk). The two alleles can be distinguished using primer pairs that specifically amplify a 360-bp wild-type fragment (5′ I0 + 3′ Ex1) or a 270-bp mutant specific fragment (5′ I0 + 3′ neo).

Figure 2

Viability of K-ras^−/− embryos. The percent recovery of viable K-ras^−/− embryos (of the total viable embryos recovered) is shown as a function of increasing gestational age. All embryos were derived from K-ras^+/− intercrosses; thus, 25% would be expected to be K-ras^−/−. This analysis was performed on both a mixed (BL/6:129/Sv) genetic background as well as on a pure 129/Sv background. The heterozygous parents used in this analysis were the F₁ progeny of chimeras bred with either pure BL/6 females (for the mixed genetic background analysis) or 129/Sv females (for the inbred genetic background analysis). On the mixed genetic background, ∼600 total implants were genotyped, ranging from 30 to 170 embryos per gestational day. On the pure 129/Sv genetic background, ∼400 total implants were genotyped. During the critical time period (E11.5–14.5), the number of embryos genotyped ranged from 30 to 130 per gestational day.

Figure 3

Phenotypic comparison of K-ras^−/− embryos and control littermates. (A) The wild-type littermate is on the left and the K-ras^−/− littermate is on the right. Note the slight developmental delay (∼0.5 gestational days) and pale coloring of the liver in the K-ras^−/− embryo. (B) The control E18.5 K-ras^+/− embryo is on the left and the K-ras^−/− embryo is on the right. Note the marked reduction in size of the mutant relative to the control littermate as well as the noncoordinate development of the K-ras^−/− embryo. The eyes have not developed beyond E15.5, whereas the limbs, tail, and skin have all advanced to at least E17.5. (C,D) Parasagittal section through an E12.5 K-ras^−/− (C) and a control wild-type (D) fetal liver. Note the areas of hypocellularity in the K-ras mutant fetal liver, whereas the cells are densely packed in the control liver. Also, pyknotic nuclei (white arrows) in the distal portion of the K-ras^−/− hepatic lobe are indicated. (E,F) Cell death analysis on an adjacent fetal liver section from C and D. Note the presence of significant numbers of TUNEL-positive (brown staining) cells in the K-ras^−/− fetal livers (E). In more severely affected embryos, these apoptotic cells were present throughout the liver. In contrast, very few cells stain positive in the TUNEL assay from control fetal livers (F).

Figure 4

Tissue contribution analysis on K−ras^+/− and K−ras^−/− chimeras. The distribution of K−ras^+/− and K−ras^−/− ES cell derivatives to various tissues was analyzed by the GPI isoenzyme assay. The percent contribution of ES cells to each tissue was estimated and categorized as follows: 0%–10% ; 11%–25% ; 26%–50% ; 51%–75% ; or 76%–100% . (ND) Not determined. K-ras^−/− ES cells had significantly reduced contribution to multiple hematopoietic lineages and the tissues that support their production throughout embryogenesis and adulthood.

Figure 5

N-Ras and H-Ras are not up-regulated in response to the loss of K-Ras. (A) MEFs were derived from E13.5 littermates on both genetic backgrounds and analyzed for the levels of K-Ras, N-Ras, and H-Ras proteins. Ras was immunoprecipitated from at least two independently derived clones for each genotype using the pan-Ras antibody Y13-259. Immunocomplexes were then analyzed by immunoblotting with monoclonal antibodies specific for each of the Ras proteins. (B) Tissues were prepared from E12.5 embryos derived on the mixed genetic background and analyzed as above. One immunoblot was made and probed with the same series of Ras antibodies as used in A. The order of analysis was as follows: N-Ras (F155), H-Ras (F235), K-Ras (F234), and then pan Ras (F111). The weak signal present in the K-ras^−/− tissues by the K-Ras-specific antibody is attributable to background cross-reactivity to the faster migrating N-ras and H-Ras proteins. Probing with the pan Ras antibody mirrored the expression levels seen with each antibody (data not shown). Similar results were also obtained with heart and lung tissues (data not shown).

Figure 6

Phenotypic comparisons of N-ras^−/−; K-ras^+/− embryos and control littermates. In situ hybridization was performed on serial parasagittal sections of wild-type E9.5 embryos using (A) K-ras, (B) N-ras, and (C) control probes. (D) RT–PCR was performed on wild-type E9.5 yolk sacs using primer pairs specific for either K-ras [lanes 2,3 (K1); lanes 4,5 (K2)] or N-ras [lanes 6,7 (N1)] with the specific products indicated at left. RNA was omitted in the control reactions (lanes 3,5,7). A 100-bp marker is represented in lane 1. (E,F) E10.5 N-ras^−/−; K-ras^+/− (left) and N-ras^+/−; K-ras^+/− (right) embryos (E) and their respective yolk sacs (F) are shown. Note that the N-ras^−/−; K-ras^+/− embryo has only developed to a stage equivalent to E9.5, and is further distinguished from its normal littermate by its dilated pericardial sac and significantly reduced numbers of circulating RBCs in either the embryo or its yolk sac. The yolk sacs also take on a roughened and wrinkled appearance compared to the smooth nature of normal yolk sacs. (G) E15.5 N-ras^−/−; K-ras^+/− embryo (left) and a control N-ras^−/−; K-ras⁺/+ littermate (right) are shown. The N-ras^−/−; K-ras^+/− embryo is developmentally delayed by 0.5 gestational days and is severely anemic and edematous. (H,I) Histological analysis of E10.5 N-ras^−/−; K-ras^+/− (H) and N-ras^+/−; K-ras^+/− (I) visceral yolk sacs. Note the strict absence of blood islands (short black arrow) and the presence of very limiting numbers of circulating primitive erythrocytes (long black arrows) in the N-ras^−/−; K-ras^+/− yolk sac. Otherwise, the yolk sac tissue and endothelial-lined blood vessels exhibit similar appearances in both yolk sacs. (J,K) Parasagittal sections through the forebrains of an E9.5 N-ras^−/−; K-ras^+/− embryo (J) and a N-ras^+/+; K-ras^+/− littermate control (K). Note the prevalent cell death [pyknotic nuclei (black arrows)] throughout the forebrain of the N-ras^−/−; K-ras^+/− embryo. This cell death extended throughout the entire embryo by E10.5. (L,M) Parasagittal sections through the fetal livers of an E15.5 N-ras^−/−; K-ras^+/− embryo (L) and a control N-ras^−/−; K-ras^+/+ littermate (M). Note the absence of blood-filled, endothelial-lined vessels (longer black arrows in M) in the N-ras^−/−; K-ras^+/− tissue. Moreover, the ratio of erythroblasts (dark, blue staining cells and smaller black arrows) to hepatocytes is significantly reduced in these embryos.