Generation of the Ceacam1a−/− mice.
The strategy leading to complete abrogation of CEACAM1a expression in mice was based on the removal of the first two exons of the Ceacam1a gene (Fig. ). The initiator ATG codon is positioned in the first exon, and many functions associated with the CEACAM1a protein depend on the presence of the first Ig-like domain encompassed within the second exon. For this purpose, an Xba1-Xho1 restriction fragment carrying these two exons was removed from the gene, and a cassette with the TK (thymidine kinase) promoter and the neor gene was inserted (Fig. ). The targeting vector was electroporated into mouse R1 ES stem cells, and chimeric mice were generated by microinjection of two ES cell lines (2D2 and 11H11) into C57BL/6 mouse blastocysts. Eight chimeric male mice were obtained, four of which transmitted the Ceacam1a+/−-targeted allele through the germ line. The heterozygous Ceacam1a+/− progeny mice were mated to produce homozygous (−/−) mice. The frequency of germ line transmission was calculated to be 22% on varied backgrounds (C57BL/6, BALB/c, and 129Sv). Mating of heterozygous mice produced expected Mendelian ratios of Ceacam1a−/− offspring (+/+, 1.0; +/−, 1.8; −/−, 0.9). CEACAM1a is expressed in ovary and prostate, yet its ablation did not alter the sex ratio of the progeny (males, 52%; females, 48%).
FIG. 1. Northern and Western analyses of Ceacam1a−/− mice. (A) The Ceacam1a gene comprises nine exons (numbered boxes) preceded by the Ceacam1a promoter (Prom.). Exon 1 contains the initiator ATG codon at the end of the 5′ untranslated (more ...) Abrogation of CEACAM1a expression in Ceacam1a-targeted mice.
The complete abrogation of CEACAM1a expression was verified by Northern and Western blotting. Total RNA was prepared from colon and liver of several mice from each litter and analyzed with formaldehyde-agarose gels. Northern blots were produced and subjected to hybridization with the 32
P-labeled full-length Ceacam1a
). The wild-type +/+ and heterozygous +/− mice produced a 4-kb RNA fragment corresponding to the Ceacam1a
transcript. The intensity of the RNA fragment in the +/− mice was approximately half of that in the control RNA produced from wild-type mice. No Ceacam1a
transcript was revealed in the homozygous −/− mice even after prolonged exposure of the membranes, indicating that the gene inactivation strategy completely abrogated Ceacam1a
transcription (Fig. ). We then examined the expression of the CEACAM1a protein isoforms in mouse colon and liver tissues by immunoblotting total proteins from several different mice with anti-CEACAM1a polyclonal Abs (Fig. ). In these and other tissues (data not shown), expression of all CEACAM1a isoforms in the homozygous −/− mice was completely eliminated relative to expression in the wild-type +/+ or heterozygous +/− mice (Fig. ). Actin protein levels in these tissues were constant (Fig. ).
Absence of CEACAM1a expression in tissues of Ceacam1a-deficient mice.
By routine histology, no histological differences were noted in the colon, small intestine, liver, kidney, prostate, ovaries, uterus, brain, lungs, heart, and spleen of −/− mice compared to those of control +/+ mice (Fig. and data not shown). Paraffin-embedded tissue sections were immunostained with anti-CEACAM1a polyclonal (Ab 2456) and monoclonal (MAb-CC1) (data not shown) Abs. When immunostained with an anti-CEACAM1a polyclonal Ab, tissues from wild-type +/+ animals exhibited strong expression on the luminal membrane of surface and crypt cells of the colon (Fig. ). Bile canaliculi of the liver and proximal tubules of the kidney were also strongly positive for CEACAM1a in +/+ mice (Fig. ). In contrast, in −/− mice, colonic (Fig. ) or intestinal (Fig. ) epithelial cells revealed no staining of the crypts, even after a longer development time. This finding was repeated for hepatocytes (Fig. ) and the collecting tubules in the kidney (Fig. ) of −/− mice. These tissues were also not immunostained with anti-CEACAM1a polyclonal Abs. Other tissues that normally express CEACAM1a (small intestine, endometrium, ovary, prostate, stomach, spleen, thymus, and lung) also displayed no immunostaining in the −/− mice (data not shown).
FIG. 2. CEACAM1a immunostaining of tissue sections from wild-type +/+ and Ceacam1a−/− mice. Paraformaldehyde-fixed colon, ileum, kidney, and liver tissues were dehydrated in ethanol and paraffin embedded. Sections of 6-μm (more ...) General health status of the offspring.
The Ceacam1a-targeted mice were viable and healthy under a pathogen-free environment. We have maintained a sizeable colony (approximately 350 individuals) of targeted mice for 1 year on the BALB/c, 129Sv, and C57BL/6 backgrounds and have not noticed any reduction in fertility, bone or cartilage abnormalities, tumors, or abnormal behavior. However, these mice do exhibit distinct phenotypes relative to liver insulin resistance and clearance (Dai et al., unpublished) and in in vivo T-cell physiology that will be reported elsewhere (R. Atallah et al., unpublished data).
Results of i.n. inoculation of Ceacam1a−/− mice with MHV-A59.
To define whether the Ceacam1a-targeted mice were susceptible or resistant to MHV infection, we subjected wild-type +/+ and −/− mice from both the 2D2 and 11H11 lines to infection with the MHV-A59 virus. Mouse litters were generated by crossing Ceacam1a+/− siblings, crossing +/+ mice with −/− mice, or mating −/− mice. Experiments were performed with the second and third backcrosses of both lines of Ceacam1a−/− mice. Genotyping was performed as described above on DNA prepared from mouse tails. Control mice were sham -inoculated, and both wild-type +/+ and −/− mice were inoculated i.n. with 10 μl of virus containing 106 PFU of MHV-A59 (Table ). The mice were examined every day for clinical signs of disease and, if warranted, were sacrificed immediately. Groups of +/+ and −/− mice were sacrificed at 4, 14, 30, 45, and 60 dpi. Blood was collected to test for Abs to viral antigens (serocoversion), and liver and spinal cord tissues were processed for pathology. Liver and brain RNA was tested for the presence of viral RNA that is indicative of viral infection.
Intranasal inoculation of Ceacamla−/− mice on C57BL/6 background with 106 PFU of MHV-A59
At 4 and 14 dpi, the health of the +/+ mice was compromised, as they showed many of the clinical symptoms described in Materials and Methods, whereas the −/− mice showed no signs of illness. Ultimately, five infected +/+ animals survived and recovered from the infection. Mice sacrificed between 30 and 60 dpi did not demonstrate overt clinical symptoms. As seen in the results shown in Table , infected +/+ mice sacrificed between 14 and 45 dpi generally exhibited high anti-virus serum Ab titers. Conversely, only two of the Ceacam1a−/− mice produced an Ab response of relatively low titer to viral antigens. Only one of four −/− mice had seroconverted at 14 dpi, none had mounted an immune response at 30 dpi or 45 dpi, and just one of five −/− mice tested weakly positive at 60 dpi. Liver histology was examined for the presence of viral lesions, and a grading system was established that took into consideration the numbers and sizes of the lesions. At early time points after infection (4 and 14 dpi) (Table ), all +/+ mice exhibited liver pathology. Indeed, +/+ mice sacrificed at 4 dpi showed lesions involving from 10% (liver pathology grade, 2+) up to more than 90% (4+) of cells (Fig. , row I). Some +/+ mice sacrificed at 14 dpi had resolving lesions affecting up to 10% (1+) of cells. After the +/+ mice recovered from signs of infection by the 30- to 60-dpi period, the liver lesions were no longer detectable. None of the −/− mice demonstrated lesions in their liver at any of the time points examined (Table and Fig. , row I).
FIG. 3. (A) Liver and spinal cord lesions in BALB/c +/+ and Ceacam1a partial knockout (p/p) mice on the BALB/c background following i.c. inoculation with 103 PFU MHV-A59. Arrows indicate areas of extensive liver destruction in wild-type +/+ (more ...)
Spinal cords from these mice were also prepared for histology and analyzed for evidence of demyelination, as indicated by loss or destruction of myelin detected by Luxol fast blue staining and the presence of asymetrical lesions (Fig. ). At 14 and 30 dpi, two of three and four of four wild-type +/+ mice, respectively, no longer had intact spinal cords and their myelin sheets were very thin, whereas none of the Ceacam1a−/− mice showed any abnormalities in these structures at any time point. Changes in spinal cord histology in the CEACAM1a-positive +/+ mice were generally correlated with the expression of viral RNA detected in the brains of affected animals. No infectious virus was detected in the brain or liver of the inoculated −/− mice, and no viral RNA from either tissue was detected by RT-PCR, even when nested PCR primer sets were used.
When Ceacam1a−/− mice were inoculated i.n. with 108 PFU of the same virus (Table ), none of the −/− mice showed clinical signs of disease or liver pathology (Fig. , row I). In contrast, livers of +/+ mice were completely devastated (liver pathology grade, 4+) upon i.n. inoculation with just 106 viral PFU. Titers of virus recovered from livers and brains of affected +/+ animals ranged from 8.4 × 102 to 4.6 × 106 PFU/g of tissue, but no infectious virus was detected in the tissues of −/− mice. These results indicate that the Ceacam1a−/− mice were completely resistant to i.n. MHV-A59 infection with at least 100-fold more than the lethal dose sustained by wild-type +/+ mice.
Intranasal inoculation of Ceacamla−/− mice on C57BL/6 background with MHV-A59a
Results of i.c. inoculation of Ceacam1a p/p mice with MHV-A59.
We had previously generated another genetically altered mouse MHVR mutant (designated p/p mice) in the BALB/c background. These animals exhibited greatly reduced expression (>95%) of the CEACAM1a receptor bearing four Ig domains while the other isoforms containing two Ig domains were overexpressed (5
). When inoculated i.n. with 106
PFU of the MHV-A59 virus, the p/p mice developed small liver lesions at 3 dpi, compared to their wild-type +/+ littermates that formed abundant, large lesions and soon succumbed to the disease. The lesions in the p/p mice completely disappeared at 7 dpi, and these animals completely recovered from the infection (5
To gauge the susceptibility of the brains and spinal cords of p/p mice to MHV-A59 without requiring prior amplification of virus inoculated at peripheral sites, we directly inoculated 103 PFU of the MHV-A59 virus i.c. and monitored the mice for the appearance of disease, altered liver and spinal cord histology, seroconversion, and production of infectious virus or viral RNA (Table ). The p/p mice showed only transient and mild signs of disease, unlike wild-type +/+ mice, which showed more-extensive neurological signs. Furthermore, 3 of 10 +/+ mice died between 4 and 14 dpi. Wild-type +/+ mice that had been inoculated i.c. and sacrificed at 4 dpi showed significantly altered liver histology (liver pathology grade, 4+) with a larger number of large lesions than the wild-type mice inoculated i.n. with the same virus. The Ceacam1a p/p mice examined at 4 dpi also exhibited liver lesions, but these were consistently fewer and smaller (2+) than those seen in the wild-type +/+ mice (Fig. row I). No demyelination of the spinal cord was observed in any mouse sacrificed at 4 dpi, but viral RNA was detected in liver and brain of all the +/+ and p/p mice. By 14 dpi, all surviving wild-type +/+ mice had medium-sized liver lesions (2+), whereas no liver lesions were found in the p/p mice. On day 14, spinal cord demyelination was observed in all wild-type +/+ mice and in three of four p/p mice, although the lesions were less extensive in p/p mice. In wild-type +/+ mice at 14 dpi, viral RNA was detected in the brains of five of five mice and the livers of three of five mice, whereas the p/p mice showed no viral RNA in liver (Table ). At 30 and 45 dpi, none of the wild-type +/+ or p/p mice exhibited liver lesions, but most (four of five +/+ and nine of nine p/p) had spinal cord demyelination (Fig. , row II). Anti-virus Abs were detected in the sera of three of three of the p/p mice sacrificed at 30 dpi and in two of two of the p/p mice sacrificed at 45 dpi. Viral RNA was detected in brains of two of three wild-type mice at day 30 and in one of one mouse sacrificed at day 45, but viral RNA was not detected in brains of p/p mice at these times. No viral RNA was detected in livers of wild-type +/+ or p/p mice at days 30 and 45 (Table ). These experiments show that MHV-A59 could infect cells in the spinal cords of p/p mice, although infection caused less-severe demyelination than in wild-type mice.
Intracerebral inoculation of Ceacamla p/p mice on BALB/c background with 103 PFU of MHV-A59
Results of i.c. inoculation of Ceacam1a−/− mice with MHV-A59.
To determine whether the virus infected the p/p mouse spinal cord via the CEACAM1a receptor, we inoculated Ceacam1a−/− mice and wild-type +/+ C57BL/6 mice with 106 PFU of MHV-A59 i.c. The results of two independent experiments are summarized in Table . Seven of 16 CEACAM1a-positive +/+ C57BL/6 mice inoculated with virus were sacrificed or died by 4 dpi and had severe liver lesions (4+). At 7 dpi, eight of the nine surviving wild-type +/+ mice were sacrificed because they showed severe signs of illness, including ataxia or paralysis. Livers showed extensive virus-induced damage (4+), and spinal cords of seven of eight mice showed demyelination. Virus was recovered from the livers and brains of the wild-type mice, with titers ranging from 102 to 105 PFU per gram of liver and 102 to 105 PFU per gram of brain. On 14 dpi, the one surviving wild-type +/+ mouse was sacrificed because it was moribund. Liver damage (2+) was less extensive than in animals at 7 dpi, but the spinal cord showed extensive demyelination.
Intracerebral inoculation of Ceacamla−/− mice on C57BL/6 background with 106 PFU of MHV-A59
In marked contrast to the lethal liver and neurological disease in wild-type +/+ mice, 36 of 36 Ceacam1a−/− mice inoculated i.c. with MHV-A59 survived (Table ). All seven −/− mice harvested at 4 dpi showed no signs of disease, had no liver or spinal cord lesions, and had neither viral RNA nor infectious virus in liver or brain. Similar results were obtained with −/− mice sacrificed at days 14, 30, and 45. Low titers of anti-virus Abs were detected in a few i.c.-inoculated −/− mice at days 7 and 45 (Table ).
These data show that 3-week-old Ceacam1a−/− mice in the C57BL/6 background are fully resistant to very high doses of the MHV-A59 virus administered i.c. or i.n. and indicate that expression of murine CEACAM1a is essential for MHV-A59 infection of liver or brain of C57BL/6 mice.