Most animals produced by somatic cell nuclear transfer (SCNT) are heteroplasmic for mitochondrial DNA (mtDNA). Oxidative phosphorylation (OXPHOS) in clones therefore requires the coordinated expression of genes encoded by the nuclear DNA and the two sources of mitochondria. Such interaction is rarely studied because most clones are generated using slaughterhouse oocytes of unrecorded origin. Here we traced the maternal lineages of seven diseased and five one-month-old live cloned piglets by sequencing their mtDNA. Additionally by using a 13K oligonucleotide microarray, we compared the expression profiles of nuclear and mtDNA-encoded genes that are involved in mitochondrial functions and regulation between the cloned groups and their age-matched controls (n=5 per group). We found that the oocytes used to generate the cloned piglets were of either the Large White or Duroc background, and oocyte genetic background was not related to the clones’ survival. Expression profiles of mtDNA-encoded genes in clones and controls showed intermixed clustering patterns without treatment or maternal lineage-dependency. In contrast, clones and controls clustered separately for their global and nuclear DNA-encoded mitochondrial genes in the lungs for both the deceased and live groups. Functional annotation of differentially expressed genes encoded by both nuclear and mtDNA revealed abnormal gene expression in the mitochondrial OXPHOS pathway in deceased clones. Among the nine differentially expressed genes of the OXPHOS pathway, seven were down-regulated in deceased clones compared to controls, suggesting deficiencies in mitochondrial functions. Together, these data demonstrate that the coordination of expression of mitochondrial genes encoded by nuclear and mtDNA is disrupted in the lung of diseased clones.

Although numerous mammalian species have been successfully cloned by somatic cell nuclear transfer (SCNT), little is known about gene expression of cloned pigs by SCNT. In the present study, expression profiles of 1-month-old cloned pigs generated from fetal fibroblasts (n = 5) were compared to those of age-matched controls (n = 5) using a 13K oligonucleotide microarray. The brain, kidney, and lung were chosen for microarray analysis to represent tissues from endoderm, mesoderm, and ectoderm in origin. In clones, 179 and 154 genes were differentially expressed in the kidney and the lung, respectively (fold change >2, p < 0.05, false discovery rate = 0.05), whereas only seven genes were differentially expressed in the brain of clones. Functional analysis of the differentially expressed genes revealed that they were enriched in diabetic nephropathy in the kidney, delayed alveologenesis as well as downregulated MAPK signaling pathways in the lung, which was accompanied with collapsed alveoli in the histological examination of the lung. To evaluate whether the gene expression anomalies are associated with changes in DNA methylation, global concentration of the methylated cytosine was measured in lung DNA by HPLC. Clones were significantly hypermethylated (5.72%) compared to the controls (4.13%). Bisulfite-pyrosequencing analyses of the promoter regions of differentially expressed genes, MYC and Period 1 (PER1), however, did not show any differences in the degree of DNA methylation between controls and clones. Together, these findings demonstrate that cloned pigs have altered gene expression that may potentially cause organ dysfunction.

The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken β‐actin/rabbit β‐globin hybrid promoter (pCAGG‐EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca 2+ followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst‐stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 ( P < 0.05). There was no significant difference in mean nuclear number. Pregnancy rate for NT2 was 100% (n = 19) at all stages collected and was significantly higher than NT1 (71.4%, n = 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n = 23; P < 0.15). Recovery rates were calculated based on the number of embryos, conceptuses, fetuses, or piglets present at the time of collection, divided by the number of embryos transferred to the recipient gilts. Recovery rates between the three groups were not significantly different at any of the stages collected ( P > 0.05). All fusion/activation treatments produced live, pCAGG‐EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer. Mol. Reprod. Dev. 76: 490–500, 2009. © 2008 Wiley‐Liss, Inc.

It is still difficult to successfully cryopreserve in vitro-produced (IVP) swine embryos, as they are sensitive to chilling due to the abundance of intracellular lipids. Mechanical delipation through micromanipulation is successful, but this method increases the potential of pathogen transmission because of the damage inflicted upon the zona pellucida during micromanipulation, and it is labor intensive. Reported here is a method to remove the lipid of IVP porcine embryos, without significantly compromising the zona pellucida, by trypsin treating the embryos or exposing the embryo to a high-osmolality solution to enlarge the perivitelline space so that the lipid could be polarized and separated completely after subsequent centrifugation without micromanipulation. The procedures work both for nuclear transfer-derived embryos and in vitro-fertilized embryos. Both methods provide a high-throughput process that leaves the zona pellucida intact (or relatively intact for the trypsin treatment) to aid in preventing disease transmission. It is also demonstrated that this procedure results in viable piglets, a claim that could not be made in many previous reports. Although the efficiencies of cryopreservation have not been dramatically improved, these procedures allow a single person to process very large numbers of embryos without the necessity of manipulating each individual embryo on a micromanipulator. Such high-throughput processing overcomes the lack of high efficiency (i.e., the system can be overloaded with embryos for transfer to surrogates).

Somatic cell nuclear transfer (SCNT) in pigs relies primarily on the utilization of fetal-derived fibroblast cells, and the resultant clones tend to exhibit a significant level of phenotypic instability, which may be due to epigenetic reprogramming and/or genomic damage in the donor cells. In addition to the compromised phenotypic stability, production of transgenic clones through SCNT is inefficient, because the restricted lifespan of somatic donor cells in culture can be limiting when the genetic modification requires selection. In contrast, stem cells proliferate rapidly and do not undergo senescence at a high rate, so the selection process can be extended. Since there is no report of an embryonic stem cell line derived in the pig that could contribute to the germ line, we decided to investigate the utility of porcine skin-derived stem cells (SSCs). Porcine SSCs were isolated from the skin on the back of day 35 to 50 Yorkshire fetuses. The SSCs were cultured continually in SSCs medium (DMEM/F12 containing B-27, 20 ng mL–1 of epidermal growth factor, and 40 ng mL–1 of basic fibroblast growth factor) at 37.8°C, 5% CO2, 95% air. The SSCs expressed the neural progenitor marker nestin, as well as genes that are critical for pluripotency, such as Oct4 and Stat3. The SSCs proliferated actively in vitro and retained a normal karyotype after long-term culture. Electron microscopy revealed 2 distinct cell types within the spheres; elongated cells at the sphere periphery had invaginated nuclear envelopes and prominent nucleoli, and these cells displayed few, but large elongated mitochondria with transversal cristae as well as large cisternae of rough endoplasmic reticulum. In contrast, the cells in the center of the spheres were predominantly round-shaped, with a large round nucleus or cuboidal. The SSCs can be genetically modified with long-term positive selection, and 50 μg mL–1 G418 appeared to be an appropriate dose of G418 for selection of the transfected SSCs. Finally, NT embryos reconstructed with SSCs showed high rates of pre- and post-implantation development.The cell number in the blastocyst stage embryos derived from cloning with the SSC was significantly higher than those of the blastocysts derived from IVF (28.5 ± 1.9, 16.8 ± 4.0, respectively, P < 0.05), although there was no significant difference in blastocyst formation rates between these groups (21 to 25%). Three of the animals became pregnant in 4 surrogate gilts which received cloned embryos and reached to term. Two healthy male cloned piglets and 1 healthy female cloned piglet are genetically identical to the SSCs. Funding for this study was provided by the National Institutes of Health.

Although transgenic animal production through somatic cell nuclear transfer (SCNT) has been successful, the process is still inefficient. One major limitation is the use of somatic donor cells that have a finite life span. Identification and isolation of a cell type capable of rapid proliferation while possessing immortal or prolonged life span in culture and is capable of being genetically modified would be very valuable for utilization in the production of genetically modified pigs. Here we report the birth of live piglets after cloning by using porcine skin-derived stem cells (SSC) as a donor cell type. In the present study, cell cycle analysis indicates that the porcine SSC proliferate rapidly in vitro. The porcine SSC are capable of producing live offspring and can be genetically modified with positive selection. Utilization of porcine SSC may prove to be an excellent cell type for genetic modification followed by nuclear transfer for the production of transgenic pigs.

Almost two decades after CFTR was identified as the gene responsible for cystic fibrosis ( CF), we still lack answers to many questions about the pathogenesis of the disease, and it remains incurable. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but the mutant mice do not develop the characteristic manifestations of human CF, including abnormalities of the pancreas, lung, intestine, liver, and other organs. Because pigs share many anatomical and physiological features with humans, we generated pigs with a targeted disruption of both CFTR alleles. Newborn pigs lacking CFTR exhibited defective chloride transport and developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn humans with CF. The pig model may provide opportunities to address persistent questions about CF pathogenesis and accelerate discovery of strategies for prevention and treatment.

We previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.

Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (Delta F508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice.

The objective of this study was to perform transcriptional profiling between in vivo (IVV), in vitro-fertilized (IVF), and nuclear transfer (NT) blastocyst stage embryos, along with the donor cell line used for NT, in order to identify candidate genes that may contribute to the suboptimal phenotypes of cloned pigs. IVV samples were collected surgically 8 days post-estrus. IVF and NT embryos were transferred into recipient gilts on Day 0 or 1 of estrus and were subsequently collected 6 days later by uterine flush. NT oocytes were activated using one of three methods:NT-1 (electrical activation/fusion), NT-2 (electrical activation/fusion + treatment with proteasomal inhibitor MG 132), or NT-3 (electrical fusion + thimerosal/dithiothreitol (DTT) activation). NT was performed by using pCAG-EGFP positive fetal fibroblast cells to avoid collection of parthenogenetic blastocysts. Donor cells were collected post-NT in pools of 100. Three pools of 10–15 embryos were collected for each treatment. Each pool was analyzed twice, resulting in three biological and two technical replicates. A reference design was used and the reference RNA represented a pool of both reproductive and non-reproductive tissues. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) and amplified by using an Ovation Ribo-SPIA linear amplification kit (NuGEN Technologies, Inc., San Carlos, CA, USA). Amplified cDNA from blastocysts or cells was labeled with Cy5 and compared to cDNA from the reference sample labeled with Cy3. The cDNAs were hybridized to an in-house printed pig reproductive tissue-specific 19 968 spot cDNA microarray. Microarray images were acquired using a GenePix 4000B scanner. Spot quality was assessed and results files were constructed using GenePix Pro 4.0. Lowess normalization and analysis was performed in Genespring 7.3.1 (Agilent Technologies, Inc., Palo Alto, CA, USA). Two comparisons were made: IVF versus IVV, and a comparison of all treatments IVV, IVF, NT-1, NT-2, NT-3, and donor cell line. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test. The IVF and IVV comparison resulted in 0 differentially detected cDNAs. The IVV, IVF, NT-1, NT-2, NT-3, and donor cell line comparison detected 1477 differentially detected cDNAs, including heat shock proteins (HSPD1 and HSPE1), which are lowly expressed in the donor cell line, and X inactive-specific transcript (XIST), which has higher expression in IVV and IVF compared to that in NT blastocysts. A standard correlation was performed on both comparisons. The R2 value for the IVV and IVF comparison was 0.892, while the R2 value for all samples was 0.716. These results illustrate that IVV and IVF blastocysts, developed within the uterus, are nearly identical. However, a comparison of blastocysts in all treatments including NT and the donor cell line revealed many differentially expressed genes that can be further evaluated for biological function and usefulness as potential markers of quality embryo development after NT.

Although transgenic animals have been successfully cloned, the process is still inefficient. One of the limitations is the use of somatic donor cells that have a limited lifespan. If a genetic modification is made, the selection process must be initiated and completed rapidly or the cells will undergo senescence. Identification of a stem cell that would proliferate rapidly and not undergo senescence would prove to be very valuable. Here we report attempts at cloning by using porcine skin-derived sphere stem cells to determine if they are a suitable donor cell type. Skin-derived stem cells were isolated from fetal skin and express the neural progenitor marker NES, as well as genes that may be critical for pluripotency such as POU5F1 and STAT3. The skin-derived stem cells proliferate rapidly in vitro and retain a normal karyotype after long-term culture. In the present study, skin-derived stem cells were cultured and frozen in liquid nitrogen from passage 1 to passage 8. To investigate the developmental potential of the skin-derived stem cells, we performed nuclear transfer (NT) and compared their preimplantation developmental efficiency to that of the embryos derived from in vitro fertilization (IVF). Cumulus–oocyte complexes (COCs) were aspirated from antral follicles of ovaries from prepubertal gilts. Approximately, groups of 50-70 COCs were matured in vitro in 500 µL TCM-199 per culture well for 40–44 h at 38.5C, in a humidified atmosphere of 5% CO2 in air. The donor cells were thawed and cultured one day before NT; skin-derived stem cells were pipetted vigorously in PBS-EDTA to isolate individual cells. For IVF, cryopreserved ejaculated spermatozoa were thawed and washed and then resuspended with fertilization medium (mTBM). The MII oocytes were co-incubated with sperm for 6 h, and then transferred to PZM3 and cultured. For NT and IVF, respectively, the percent cleavage at 48 h in PZM3 was 64.9 8.2% (169/208) and 62.1 3.1% (94/184) (P > 0.05), the percent blastocysts after 6 days was 21.5 5.8% (53/208) and 25.2 3.4% (46/184) (P > 0.05), and the number of nuclei per blastocyst was 28.5 1.9 (NT, maximum was 58) and 16.8 4.0 (IVF, maximum was 31) (P < 0.05). To determine development post-implantation, some cloned embryos were cultured in PZM3 for 15.5 h and an average of 112 cloned embryos were transferred to the oviducts of four naturally cycling gilts on Day 0–1 of standing estrus. Three of the animals were pregnant: one of them farrowed two male piglets on August 14th, with the other two due on September 8th and 9th. Future studies will involve performing NT and ET on skin-derived stem cells from a higher passage number to determine if they would be suitable for genetic modification prior to NT.

An optimal environment for fertilization and early embryonic development is provided by the mammalian oviduct and uterus. The secretory cells lining the lumen of the oviduct and uterus synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. Western blotting in this study demonstrated that a 50‐kDa secreted phosphoprotein 1 (SPP1) form was present in the uterus on Days 0, 3, and 5 in pregnant and nonbred gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5 in pregnant gilts, but in nonbred gilts the concentration of SPP1 on Day 0 was higher than Day 3, but not Day 5. In addition, we show that addition of 0.1 µg/ml SPP1 to the culture medium after fertilization increased the percent cleaved (24 hr: 23.6 ± 1.29a vs. 18.7 ± 0.65b (2‐cell %)), and the percent blastocyst (37.2 ± 1.12a vs. 30.9 ± 0.56b) derived from IVF (P < 0.05). In parthenogenetic‐derived embryos the percent cleaved was increased due to SPP1 at 24 hr (24.0 ± 1.59a vs. 19.7 ± 1.59b (>2‐cell %)), and at 48 hr (72.9± 2.99a vs. 63.3 ± 2.99b), but not the percent blastocyst. By TUNEL assay, SPP1 decreased both apoptosis (7.9 ± 0.04a vs. 13.1 ± 0.02b) and the percent fragmentation (45.2 ± 0.07a vs. 58.8 ± 0.03b). We conclude that SPP1 can improve development in vitro possibly by reducing the rate of apoptosis. Mol. Reprod. Dev. 75: 291–298, 2008. © 2007 Wiley‐Liss, Inc.

Osteopontin (SPP1) is a multifunctional phosphoprotein (50 kDa in the pig) that has been identified in the fluid of porcine oviduct and uterus, and can be localized to the ampulla of the oviductal epithelium. Previous studies revealed that SPP1 increased the fertilization efficiency during porcine in vitro fertilization (IVF), and improved in vitro development of porcine early embryos by reducing apoptosis. Here we investigated the effect of SPP1 on in vitro maturation (IVM) and in vitro development, as well as whether SPP1 acts through the activation of the phosphatidylinositol 3-Kinase/Akt signaling pathway (PI3K). Cumulus oocyte complexes were cultured for 42–44 h in IVM medium (TCM199 supplemented with PVA, glucose, sodium pyruvate, cysteine, LH, FSH, EGF). At 18 hours after IVM, the oocytes were distributed to eight groups with and then cultured for 24 hours. The treatments were: Control; 0.1 μg/ml SPP1; 40 μM LY294002 (hereafter called LY) in DMSO; 80 μM LY in DMSO; 0.1 μg/ml SPP1 + 80 μMLY in DMSO (pretreated for 2 h with LY); DMSO; 0.1 μg/ml IL-3; 0.1 μg/ml IL-3 (pretreated for 2 h with LY). The intensity of cumulus expansion was classified as fully expanded cumulus, moderately expanded cumulus, or poorly expanded cumulus. The MII oocytes were selected and fertilized. At 6 hours after IVF, the embryos were transferred to PZM-3 supplemented with 3 mg/ml BSA. The maturation rate, cleavage rate at 24 h and 48 h, blastocyst rate at days 6 and 8, arrested embryos rate, and fragmented embryos rate were determined. The results show that the intensity of cumulus expansion was influenced by SSP1 and inhibitor LY: the full expansion rate in the groups with LY was significantly lower than control and the groups without LY294002: 10.8 % (40 μM LY), 8.8% (80 μM LY), 2.0% (SPP1+LY), 14.1% (IL-3+ LY), 21.55% (control), 33.4% (SPP1), 36.16% (IL-3), 24.1% (DMSO). The percentage maturation in the groups with LY was significantly lower than control and the groups without inhibitor: 20.4 % (40 μM LY), 10.5% (80 μM LY), 31.0% (SPP1+LY), 24.4% (IL-3+LY), 53.8% (control), 65.3% (SPP1), 69.3% (IL-3), 65.0% (DMSO). The cleavage rate at 24 h and 48 h were not different. The percent blastocyst was not significantly different except that in LY group (at day 6: 10.6% (40 μM), 7.8% (80 μM), 10.6% SPP1+LY), 11.8% (IL-3+LY), 13.5% (control), 20.55% (SPP1), 11.3% (IL- 3), 20.7% (DMSO); at day 8: 8.3% (40 μM LY), 0% (80 μM LY), 11.5% (SPP1+LY), 10.7% (IL-3+LY), 10.2% (control), 20.4% (SPP1), 10.2% (IL-3), 16.6% (DMSO). The cell number per blastocyst in LY groups was significantly less than other groups: 10.5 (40 μM LY), 0 (80 μM LY), 47.4 (SPP1+LY), 35.3 (IL-3+ LY), 25.4 (control), 27.9 (SPP1), 33.0 (IL-3), 35.3 (DMSO). This study found that cumulus expansion was affected by SPP1, IL-3, DMSO and LY. The poor expansion with inhibitor leads to the lower maturation rate. The cleavage rate in the LY groups was not decreased, but further embryonic development was reduced. This study demonstrated that SPP1 affected the maturation of porcine oocytes involving in PI3K signal pathway. Funded in part by Food for the 21st Century. (poster)

The objective of this study was to identify abnormally expressed genes early in gestation that may contribute to the suboptimal phenotypes of cloned and genetically modified pigs. Differential expression patterns were evaluated between in vivo (IVV), in vitro produced (IVP) and nuclear transfer (NT) derived extraembryonic membranes (EM) at day 30 of gestation using microarrays. IVV samples were collected via surgery of bred gilts. IVP and NT embryos were transferred into recipient gilts on day 0 or 1 of estrus. Gilts were subsequently euthanized on day 30 of pregnancy. Fetal tissue and EM were separated and snap frozen. NT oocytes were activated using three different methods, NT-1 (electrical activation/fusion), NT-2 (electrical activation/fusion + treatment with proteasomal inhibitor MG 132) and NT-3 (electrical fusion + Thimerosal/DTT activation). NT was performed using pCAG-EGFP positive fetal fibroblast cells to avoid accidental collection of parthenogenetic tissue. Additionally, IVV and IVP samples and the fetal fibroblasts used for NT were all derived from the same sire to decrease sire-specific variation. Three pigs represent each treatment group and each EM was analyzed twice, resulting in three biological replicates and two technical replicates. A reference design was used and the reference RNA represented pool of both reproductive and non reproductive tissues. Total RNA was isolated from EM and reference samples using RNA Stat 60 (Tel Test, Inc.). Fifteen micrograms of RNA was converted to cDNA using a SuperScript Indirect cDNA Labeling System (Invitrogen). cDNA from EM was labeled with cy5 and compared to cDNA from the reference sample labeled with cy3. Labeled cDNA was hybridized to our in-house printed pig reproductive tissue-specific 19,968 spot cDNA microarrays. Microarray images were acquired using a Genepix 4000B scanner. Spot quality was assessed and results files were constructed using Genepix Pro 4.0. Results files from only the good spots were loaded into Genespring 7.2 for analysis and Lowess normalization was performed. Two comparisons were made: IVP versus IVV and a comparison of all treatments IVV, IVP, NT-1, NT-2, and NT-3. ANOVA (p<0.05) was performed with the Benjamini and Hochberg False Discovery Rate multiple correction test. The IVP and IVV comparison resulted in 0 differentially detected cDNAs. The IVV, IVP, NT-1, NT-2, and NT-3 comparison differentially detected 3338 cDNAs. A standard correlation was performed on both comparisons. The R2 value for the IVV and IVP comparison was 0.819 while the R2 value for all samples was 0.759. These results illustrate that in the pig, the IVV and IVP extraembryonic membranes at day 30 of gestation are quite similar and have no differentially expressed genes. However, a comparison of EM in all treatments including IVV, IVP and NT-1, NT-2 and NT-3 resulted in many differentially expressed genes at the same stage. (platform)

Background: This prospective observational study compared endtidal carbon dioxide (PECO2) with blood gas carbon dioxide (PaCO2) values in children sedated by nonanesthesiologists for cardiac catheterization.Methods: A nasal cannula designed to obtain gas sampling simultaneously from over the mouth and nares was taped into place after assuring a good waveform. Patients' cardiac lesions, site of blood gas sampling and P(E)CO(2)were recorded.Results: Two hundred and one blood/PECO2 pairs were measured in 59 patients from 4 days to 18 years of age. Linear regression, Pearson correlation, and Bland-Altman analysis revealed a reasonable relationship (r = 0.493, P < 0.01, bivariate Pearson correlation) for all blood/expired CO2 pairs even when the blood sample was obtained from an area of the circulation with shunting. There was no significant difference in the accuracy of the blood/PECO2 pairs between infants who weighed <= 15 kg compared with children who weighed > 15 kg. Thirteen children were diagnosed with partial or complete airway obstruction.Conclusions: Endtidal CO2 measurement provides a reasonable reflection of blood CO2 values if the expired gas-sampling catheter is taped in place after assuring a good waveform. The veracity of the data was the same throughout the patient size range. Expired CO2 monitoring is useful for assessing the adequacy of respirations and the patency of the airway in children 3-89 kg.

To the Editor: Mencke et al. (1) selected a relatively low dose of rocuronium (0.6 mg/kg) in their prospective study relating airway injury to choice of neuromuscular blocking drug. There is substantial evidence (cited within the study background) that 0.6 mg/kg of rocuronium produces inferior intubating conditions compared to succinylcholine for rapid-sequence induction/intubation, especially when thiopental is used as an induction drug. I question whether this study design provides clinically relevant information. Because most practitioners use a higher dose of rocuronium for rapid-sequence intubation, the dose used in this study may not represent typical or appropriate dosing. Also, if the authors’ prior study (2) (saline versus atracurium) had already shown increased airway injury with inferior intubating conditions, it might be argued that this follow-up study was really just an indirect comparison of intubating conditions between saline after 3 min and low-dose rocuronium after 50 s. But again, the clinical relevance of an unrealistic rocuronium dose/timing combination is unclear. Another interesting aspect of this study is an ethical one. Because the authors had already observed increased injury with inferior intubating conditions in their prior study, what was the rationale here for subjecting another group of patients to similar conditions and therefore increased risk of airway injury? Moreover, the subject group they chose was composed of patients at increased risk for pulmonary aspiration. To maximize safety in both clinical practice and research, such patients require preparation with the best intubating conditions possible to achieve rapid and successful intubation. Deliberately creating inferior intubating conditions in such an at-risk patient population appears imprudent. So I am left wondering how the authors justify a study design that subjects patients to an increased risk of airway injury and aspiration pneumonitis to answer what may be a clinically irrelevant question. Similarly, I wonder why patients, if fully informed, would consent to participate in such a study. David B. Wax, MD Mount Sinai School of Medicine New York, New York [email protected]