KRAS mutation leads to decreased expression of regulator of calcineurin 2, resulting in tumor proliferation in colorectal cancer.

KRAS mutations occur in 30-40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apc(flox/flox); LSL-Kras(G12D) and CDX2P-G22Cre;Apc(flox/flox) mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT-PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin-nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin-NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC.

Developmental ability of trophoblast stem cells in uniparental mouse embryos.

Neither parthenogenetic (PG) nor androgenetic (AG) mouse embryos survive after day 9.5 of pregnancy, owing to the inadequate growth of extraembryonic tissues, including the placenta. At day 9.5 of pregnancy, the placental structures are poorly developed in PG embryos, while trophoblast giant cells are abundant at the implantation site in AG embryos. These findings suggest that both parental genomes are required for placental development. To gain further insight into the trophoblast lineage in PG and AG embryos, we attempted to derive trophoblast stem (TS)-like cell lines from uniparental embryos. Furthermore, we sought to assess their ability to differentiate into cells of the trophoblast lineage by using gene expression analysis. Three cell lines that expressed marker genes for undifferentiated TS cells (Cdx2 and Errbeta) were derived from AG embryos. Under differentiation conditions, these cells expressed the trophoblast giant cell-specific genes, but did not express the spongiotrophoblast-specific genes. In contrast, none of the four cell lines from PG embryos expressed marker genes for undifferentiated TS cells, but they expressed Oct3/4, a marker gene for embryonic stem cells. Immunohistochemical analysis indicated that PG blastocysts expressed Oct3/4 and Cdx2 specifically in inner cell mass and the trophectoderm respectively. These results suggest that PG embryos do not possess TS cells, because of the lack of the developmental ability of trophoblast cells.

Disruption of imprinting in cloned mouse fetuses from embryonic stem cells.

Cloned mice typically display abnormal development, such as overgrowth of fetuses and placentae. Quantitative expression analysis of eight imprinted genes (H19, Igf2, Igf2r, Air, Peg1/Mest, Peg3, Nuronatin (Nnat) and Ndn) and an alternate transcript of Igf2 (P0) in embryonic stem cloned fetuses and placentae at days 9.5, 12.5 and 17.5 after mating was carried out by real time PCR to investigate whether epigenetic modification of imprinted genes is responsible for overgrowth of the fetus and placental hypertrophy. In addition, the methylation pattern through the bisulphite sequencing method in differentially methylated regions of H19 and Igf2r was examined in day 9.5 fetuses and placentae. The results showed clearly that the expression of H19 gene decreased in cloned fetuses at days 12.5 and 17.5 after mating and in placentae at day 17.5 after mating, and Igf2 was also repressed in fetuses at days 9.5 and 12.5 after mating and in placentae at day 17.5 after mating. In contrast, the transcription of P0, which is a placental-specific transcript variant of Igf2, increased at more than four times the control in cloned placenta at day 12.5 after mating. Day 9.5 fetuses that have developed normally revealed only hypermethylated alleles in the H19 differently methylated region (DMR), and both hyper- and hypomethylated alleles in the Igf2r DMR2. These results show that inappropriate reprogramming in some imprinted genes affects the development of cloned embryos, and that aberrant P0 Igf2 transcription in particular may cause the overgrowth of cloned fetuses and placentae.

Identification of a new imprinted gene, Rian, on mouse chromosome 12 by fluorescent differential display screening.

Systematic screening of differentially expressed genes among androgenetic, parthenogenetic, and normal embryos by means of fluorescent differential display revealed five imprinted genes. One of them, named Rian, was expressed exclusively from the maternal allele and was closely linked to an imprinted gene, Meg3(Gtl2), mapped to the distal end of chromosome 12. The Rian transcript did not have any apparent open reading frame, and its transcript was exclusively localized to the nucleus.

Disruption of imprinted expression of U2afbp-rs/U2af1-rs1 gene in mouse parthenogenetic fetuses.

The present study shows that the U2afbp-rs gene, a paternally expressed imprinted gene, is activated and expressed in a biallelic manner from maternal alleles in parthenogenetic mouse fetuses on day 9.5 of gestation. The mean expression was detected to be 88% (31-134%) of that in control biparental fetuses, using real-time quantitative reverse transcription and polymerase chain reaction. This disrupted expression of the gene was associated with changes in the chromatin structure but not with the methylation pattern in the regulation region. The present results show that parental specific expression of imprinted genes is not always maintained in uniparental embryos. This suggests that both parental genomes are necessary to establish parental specific expression of the U2afbp-rs gene.

Efficient cryopreservation of bovine blastocysts derived from nuclear transfer with somatic cells using partial dehydration and vitrification.

Preservation by vitrification of Day 7 and Day 8 bovine blastocysts derived from nuclear transfer with cumulus cells was compared with preservation of in vitro fertilized blastocysts. In Experiment 1, embryos were vitrified in PBS containing 60% ethylene glycol. In Experiment 2, they were vitrified in combination with partial dehydration using a solution of 39% ethylene glycol + 0.7 M sucrose and 8.6% Ficoll. In Experiment 1, survival and hatching rates were 44 and 95% for nuclear transferred embryos, and 78 and 55% for in vitro fertilized embryos, respectively. In Experiment 2, survival and hatching rates were 93 and 95% for nuclear transfer embryos, and 77 and 85% for in vitro fertilized embryos, respectively. It is concluded that Day 7 and Day 8 bovine blastocysts derived from cumulus cells could be cryopreserved without the loss of viability by a simple and efficient method using a combination of partial dehydration and vitrification.

Induction of pluripotency by injection of mouse trophectoderm cell nuclei into blastocysts following transplantation into enucleated oocytes.

Pluripotency of mouse trophectoderm (TE) cells was examined using a nuclear transfer technique. We transferred a TE cell to an enucleated oocyte and cultured the reconstituted oocyte to be blastocyst stage. Then a portion of the inner cell mass (ICM) isolated from the TE-origin blastocyst was injected into the cavity of a fertilized blastocyst to produce a chimeric embryo, which was transferred to a recipient female. Of 319 oocytes reconstituted with TE cells, 263 (82.4%) had a single nucleus (1PN), 3 (0.9%) had 2 nuclei (2PN) and 53 (16.6%) had a nucleus with a polar body (1PN1PB). Although the oocytes with 1PN and 2PN developed to blastocysts (81 of 263, 30.8% and 1 of 3, respectively), only those with 1PN were used to produce chimeric blastocysts. After the transfer of chimeric embryos to recipient females, 7 (28%) of 25 conceptuses analyzed at midgestation showed chimerism. Of those 5 (71%), 6 (86%) and 4 (57%) chimeric conceptuses showed distribution of donor nuclei in the fetus, membrane and placenta, and the distributions were 10 to 65, 10 to 50 and 10 to 15%, respectively. Of the 23 young obtained, 7 (30%; 2 males and 5 females) were coat color chimeras. The contributions of donor nuclei were detected in the brain, lung, heart, liver, kidney, testis, ovary and blood. Each coat-color chimeric mouse was mated with CD-1 male or female mice, but no germ line chimera was obtained. When ICM cells were used as the control nuclear donor, the contribution was equivalent to those of TE cells. In conclusion, pluripotency of mouse TE cells on a somatic line was induced, and chimeric young were obtained using a nuclear transplantation technique.

Eight calves cloned from somatic cells of a single adult.

Eight calves were derived from differentiated cells of a single adult cow, five from cumulus cells and three from oviductal cells out of 10 embryos transferred to surrogate cows (80 percent success). All calves were visibly normal, but four died at or soon after birth from environmental causes, and postmortem analysis revealed no abnormality. These results show that bovine cumulus and oviductal epithelial cells of the adult have the genetic content to direct the development of newborn calves.

Production of monozygotic twins after freezing and thawing of bisected mouse embryos.

We examined the viability of mouse bisected embryos after freezing and thawing and produced monozygotic twin mice from these embryos. Two-cell embryos were collected from superovulated mature agouti, F1 hybrid (C57BL/6 x CBA) female mice. For bisection, one blastomere of the embryo was aspirated with a micropipette and injected into an empty zona pellucida. After culture for 24 to 28 h to the compacted 4- to 8-cell stage or 48 to 52 h to the late morula to blastocyst stage, the embryos were slowly frozen (-0.5 to -1.0 degrees C/min), thawed (30 degrees C/min), cultured for 24 h, and then transferred to recipient females. The bisected embryos without zonae pellucidae had developmental ability in vitro similar to those with zonae pellucidae (88% vs 89%). However, after freezing and thawing at the compacted 4- to 8-cell stage, bisected embryos with zonae pellucidae had higher viability than those without (60% vs 15%). Zona enclosed, bisected embryos frozen at the compacted 4- to 8-cell stage were more resistnat to freezing and thawing than those at the late morula to blastocyte stage (60% vs 23%). After transfer to recipients 26% of the zona enclosed bisected embryos frozen-thawed at the 4- to 8-cell stage developed to living fetuses a day 17.5 to 18.0 of pregnancy, which was slightly but not significantly lower than that of fresh bisected embryos (48%). On the other hand, only 5% of bisected embryos frozen-thawed at the late morula to blastocyst stage developed to young. The transfer of 15 sets of twin blastocysts as pairs that had been frozen and thawed at the compacted 4- to 8-cell stage yielded 2 (13%) sets of monozygotic twins.

A comparative investigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocysts to produce chimeras.

The ability of trophectoderm (TE) cells to produce chimeric mice (pluripotency) was compared with that of inner cell mass (ICM) cells. TE and ICM cells of blastocysts and hatching or hatched blastocysts derived from albino mice (CD-1, Gpi-1a/a) were aggregated with zona cut 8- to 16-cell stage embryos or injected into the blastocoele from non-albino mice (C57BL/6 x C3H/He, Gpi-1b/b). After transfer to pseudopregnant female mice, the contribution of the donor cells was examined by glucose phosphate isomerase (GPI) analysis of embryos, membrane and placenta at mid-gestation (Day 10.5 and 12.5) or by the coat color of newborn mice. In contrast to ICM cells, there was no contribution of TE cells in the conceptuses and no coat color chimeric young were obtained. After pre-labeling of TE cells with fluorescent latex microparticles, they were aggregated with embryos and the allocation of TE cells at the compacted morula and blastocyst stages was observed under a fluorescent microscope. Although the TE cells were observed attached onto the surface of the embryos at morula and blastocyst stages, unlike the ICM cells, they were not positively incorporated into the embryos. Thus, the pluripotency of TE cells from mouse blastocysts was not induced by the aggregation and injection methods.

Effect of ooplast activation on the development of oocytes following nucleus transfer in cattle.

We assessed the effect of ooplast (enucleated oocytes) activation prior to receiving a donor nucleus on the development of nucleus transferred oocytes in cattle. The ooplasts were activated by electric stimulus at 30, 33, 36 and 39 h after being placed in culture medium for meiotic maturation. The activated ooplasts were further cultured in vitro, for a total 42 h from the beginning of maturation, 16- to 32-cell stage embryos produced by in vitro fertilization were used as donor embryos. The nucleus transferred oocytes were co-cultured with bovine oviductal epithelial cells in vitro. The fusion rate was not different between the activated (90%) and aged (94%) ooplasts 42 h after culture. Activated ooplasts receiving a donor nucleus showed a higher developmental rate than the aged ooplasts. Maximal development of the oocytes was obtained if the ooplast was activated at 9 h prior to receiving a donor nucleus. Thirty-nine percent developed to morulae and 24% to blastocysts. This compares (P<0.01) with 13% of the aged ooplasts developing to morulae and 8% to blastocysts. Of the activated ooplasts at 3, 6 and 12 h prior to fusion with a donor blastomere, 12, 16 and 13% developed to blastocysts, respectively. Of the 17 recipient cows receiving nucleus transferred embryos, 9 (53%) were diagnosed pregnant by palpation per rectum examination, and 3 normal offspring were obtained.

Development of androgenetic mouse embryos produced by in vitro fertilization of enucleated oocytes.

Enucleated mouse oocytes were successfully fertilized in vitro, and the resultant androgenetic eggs developed to the blastocyst stage. The proportion of enucleated oocytes fertilized in vitro was high (87-99%) at sperm concentrations ranging from 10-100 x 10(4)/ml. At high sperm concentrations (100-1,000 x 10(4)), 35-45% of the fertilized eggs resulted in heterozygous bispermic androgenones. The proportion of hemizygous haploid and heterozygous diploid androgenones developing to blastocysts was 11% and 43%, respectively. Hemizygous diploidization, however, showed no positive effect on development. These results clearly show that the procedure reported here is efficient and reliable for the production of androgenetic eggs.