Targeted Inactivation of p12Cdk2ap1, CDK2 Associating Protein 1, Leads to Early Embryonic Lethality

Targeted disruption of murine Cdk2ap1, an inhibitor of CDK2 function and hence G1/S transition, results in the embryonic lethality with a high penetration rate. Detailed timed pregnancy analysis of embryos showed that the lethality occurred between embryonic day 3.5 pc and 5.5 pc, a period of implantation and early development of implanted embryos. Two homozygous knockout mice that survived to term showed identical craniofacial defect, including a short snout and a round forehead. Examination of craniofacial morphology by measuring Snout Length (SL) vs. Face Width (FW) showed that the Cdk2ap1+/− mice were born with a reduced SL/FW ratio compared to the Cdk2ap1+/+ and the reduction was more pronounced in Cdk2ap1−/− mice. A transgenic rescue of the lethality was attempted by crossing Cdk2ap1+/− animals with K14-Cdk2ap1 transgenic mice. Resulting Cdk2ap1+/−:K14-Cdk2ap1 transgenic mice showed an improved incidence of full term animals (16.7% from 0.5%) on a Cdk2ap1−/− background. Transgenic expression of Cdk2ap1 in Cdk2ap1−/−:K14-Cdk2ap1 animals restored SL/FW ratio to the level of Cdk2ap1+/−:K14-Cdk2ap1 mice, but not to that of the Cdk2ap1+/+:K14-Cdk2ap1 mice. Teratoma formation analysis using mESCs showed an abrogated in vivo pluripotency of Cdk2ap1−/− mESCs towards a restricted mesoderm lineage specification. This study demonstrates that Cdk2ap1 plays an essential role in the early stage of embryogenesis and has a potential role during craniofacial morphogenesis

Latitude variation in pancreatic cancer mortality in Australia

Objectives: Ecological studies support the hypothesis that there is an association between vitamin D and pancreatic cancer (PaCa) mortality, but observational studies are somewhat conflicting. We sought to contribute further data to this issue by analyzing the differences in PaCa mortality across the eastern states of Australia and investigating if there is a role of vitamin D-effective ultraviolet radiation (DUVR), which is related to latitude. Methods: Mortality data from 1968 to 2005 were sourced from the Australian General Record of Incidence and Mortality books. Negative binomial models were fitted to calculate the association between state and PaCa mortality. Clear sky monthly DUVR in each capital city was also modeled. Results: Mortality from PaCa was 10% higher in southern states than in Queensland, with those in Victoria recording the highest mortality risk (relative risk, 1.13; 95% confidence interval, 1.09-1.17). We found a highly significant association between DUVR and PaCa mortality, with an estimated 1.5% decrease in the risk per 10-kJ/m2 increase in yearly DUVR. Conclusions: These data show an association between latitude, DUVR, and PaCa mortality. Although this study cannot be used to infer causality, it supports the need for further investigations of a possible role of vitamin D in PaCa etiology

Vitamin D intake, sun exposure and 25-hydroxy vitamin D status in Peritoneal dialysis (PD) and Haemodialysis (HD) patients

Inadequate vitamin D levels have been linked to bone disease but more recently have been associated with wider health implications. Limited studies suggest a high prevalence of Vitamin D deficiency in dialysis patients, although evidence is lacking on whether this is due to dietary restrictions, limited mobility and time outdoors or a combination of these. The aim of this study was to assess the contributions of diet, supplements and sunlight exposure to serum Vitamin D (25(OH)D) levels in dialysis patients. Cross-sectional data were obtained from 30 PD (Mean±SD age 56.9±16.2 y; n=13 male) and 22 HD (Mean±SD age 65.4±14.0 y; n=18 male) patients between 2009 and 2010. Serum 25(OH)D was measured and oral vitamin D intake estimated through a food-frequency-questionnaire and quantifying inactive supplementation. Sunlight exposure was assessed using a validated questionnaire. Prevalence of inadequate/insufficient vitamin D differed between dialysis modality (31% and 43% insufficient (<50nmol/L); 4% and 34% deficient (<25nmol/L) in HD and PD patients respectively (p=0.002)). In HD patients, there was a significant correlation between diet plus supplemental vitamin D intake and 25(OH)D (ρ=0.84, p<0.001). Results suggest a higher frequency of 25(OH)D inadequacy/deficiency in PD compared to HD patients. No other relationships between intake, sun exposure and 25(OH)D were seen. This could reflect limitations of the study design or the importance of other factors such as age, ethnicity and sun protection as interactions in the analysis. Understanding these factors is important given Vitamin D’s emerging status as a biomarker of systemic ill health

Transgenic rescue result from genotyping of <i>Cdk2ap1^{+/−:K14-Cdk2ap1}</i> mating.

<p>Transgenic rescue result from genotyping of <i>Cdk2ap1^{+/−:K14-Cdk2ap1}</i> mating.</p

Timed pregnancy analysis by Het x Het matings.

<p>Timed pregnancy analysis by Het x Het matings.</p

Generation of <i>Cdk2ap1</i> knockout mice.

<p>A. Targeting vector construct <i>pTKLNCL-Cdk2ap1</i> was designed to knockout <i>Cdk2ap1</i> exon2 and 3 by homologous recombination. Cytosine deaminase (CD) and neomycin resistance (<i>Neo</i>) genes were placed in to disrupt <i>Cdk2ap1</i> gene in mouse chromosome 5. B. Multiplex genotyping analysis was used to screen recombinant ES cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004518#s2" target="_blank">Materials and Methods</a>. The top band of 460 bp is amplified from cytosine deaminase in the targeting vector and the bottom band of 239 bp is amplified from the introns of wild type <i>Cdk2ap1</i>. Mouse tail DNA was also analyzed for screening of <i>Cdk2ap1</i> knockout mice by multiplex PCR. C. The multiplex PCR genotyping data was confirmed by Southern blot analysis. The wild type <i>Cdk2ap1</i> showed 8.4 kb fragment, while the recombinant showed 5.6 kb fragment.</p

Genotyping and expression analysis of <i>Cdk2ap1^{−/−:K14-Cdk2ap1}</i> hybrids.

<p>Hybrid mice were generated by crossing heterozygous <i>Cdk2ap1</i> knockout mice and <i>K14-Cdk2ap1</i> transgenic mice. A. The resulting pups were genotyped to identify hybrids by using PCR. Wild type <i>Cdk2ap1</i> gene was amplified by using Int4F and IntR primers against the <i>Cdk2ap1</i> intron sequence. The recombinant <i>Cdk2ap1</i> was detected by using CD3F and CD3R primers against cytosine deaminase gene. In addition, the presence of transgenic <i>Cdk2ap1</i> was detected by using <i>hK14</i> and <i>mCdk2ap1</i> primers against human K14 promoter and mouse <i>Cdk2ap1</i> transgene. By intercrossing heterozygous <i>Cdk2ap1^+/−:K14-Cdk2ap1</i> mice, we were able to generate homozygous <i>Cdk2ap1^−/−:K14-Cdk2ap1</i> mice. Mouse ESCs (<i>Cdk2ap1^+/+</i>, <i>Cdk2ap1^+/−</i>, and <i>Cdk2ap1^−/−</i>) and pups (WT and heterozygous) from mating of heterozygous <i>Cdk2ap1</i> knockout mice were used as controls. B. The expression of <i>Cdk2ap1</i> was examined by RT-PCR analysis on total RNA from earsnips as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004518#s2" target="_blank">Materials and Methods</a>. Compared to no RT or <i>Cdk2ap1^−/−</i> mESC control, comparable amount of <i>Cdk2ap1</i> mRNA was detected in <i>Cdk2ap1^−/−:K14-Cdk2ap1</i> mouse tissues, which showed that the expression of <i>Cdk2ap1</i> was achieved from the <i>Cdk2ap1</i> transgene in the mice with <i>Cdk2ap1^−/−</i> genotype.</p

<i>Cdk2ap1^−/−</i> mESCs showed an abrogated <i>in vivo</i> pluripotency.

<p><i>In vivo</i> pluripotential competence of <i>Cdk2ap1^−/−</i> mESCs was evaluated by teratoma formation analysis. <i>Cdk2ap1^+/+</i> or <i>Cdk2ap1^−/−</i> mESCs were transplanted into the testis of SCID mice in duplicate as described by Conway et al. (29). After 4 weeks, tumors were extracted and subjected to fixation and sectioning. The slides were stained with H&E and examined under bright field microscope. A. Gross examination of teratoma sections from <i>Cdk2ap1^+/+</i> and <i>Cdk2ap1^−/−</i> mESCs (×4 magnification). B. Specified three lineages committed from <i>Cdk2ap1^+/+</i> mESCs. C. A restricted commitment of <i>Cdk2ap1^−/−</i> mESCs to a certain mesoderm lineage.</p

Blastocyst outgrowth analysis of <i>Cdk2ap1</i> knockout embryos.

<p>To examine the hatching and outgrowth of <i>Cdk2ap1^−/−</i> embryos, blastocyst outgrowth analysis was performed. Heterozygous females and males were mated for timed pregnancy analysis and E3.5 blastocysts were collected. A. Individual blastocyst was cultured in a 96 well plate for 4 days. Photomicrograph was taken every day to monitor the hatching and outgrowth of blastocysts. B. After final recording of morphology, outgrown cells were lysed and genomic DNA was isolated for multiplex genotyping analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004518#s2" target="_blank">Materials and Methods</a>. The PCR products were analyzed on a 1.5% agarose gel and the genotype was matched to the phenotype from A.</p