Search

Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P

The Y chromosome–linked gene sex-determining region (SRY) is believed to be the master initiator of male sex determination in almost all eutherian mammals, functioning to upregulate expression of its direct target gene SRY-related HMG box–containing gene 9 (SOX9). XX maleness in humans is a rare syndrome with a frequency of 1 in 20,000–25,000 males. Approximately 90% of these cases carry some amount of the Y chromosome sequences due to an illegitimate recombination between X and Y chromosomes, where most of these cases have normal male genitalia. A more severe manifestation, the remaining 10% do not have any Y-chromosome sequences, and typically have ambiguous genitalia. In this study, we report a case of an infertile bull. Physical examination of this purebred Holstein bull showed a phenotypically androgynous animal with a normal penis, cryptorchidism (2 diminutive testicles, only one descended), and with a skeletal conformation and stance usually associated with female individuals. Lymphocytes from heparin sodium anticoagulated blood were cultured, harvested, and prepared according to conventional cell culture methods revealed a normal 60, XX karyotype with no numerical or structural chromosomal aberrations. PCR analyses were negative for the SRY gene, and positive for androgen receptor gene on the X chromosome. No sperm were found in seminal fluid collected in a routine breeding soundness exam at 17 months of age. The bull was slaughtered at 18 months of age, and its reproductive tract was collected. The testicle weighed 18.9 g (descended), and 42.4 g (abdominal). Neither testicle/epididymal unit was appropriate size or fully developed. Two very enlarged vesicular glands (43.36 g and 52.52 g, normally each ~ 20 g) were observed alongside what appeared to be uterine tissue. Histology showed no tubular grooves in the ampulla and an epididymis devoid of sperm. The development of a male phenotype in SRY-negative XX individuals suggests that in addition to SRY, there are autosomal or X-linked genes involved in the sex-determining pathway. In humans, a dose change of the SOX9 gene, loss of function mutations in the WNT4, FoxL2, and RSPO1 testicular suppressing genes, and genomic rearrangements within the regulatory region of the SRY-box transcription factor 3 (SOX3) gene have all been associated with XX male sex reversal. To investigate whether a mutation or genomic rearrangement was responsible for the male phenotype in the SRY-negative 60, XX bull, DNA was collected from the bull and his sire and dam for long-read trio sequencing. These data may provide evidence for either a loss of function mutation in a gene normally inhibiting testes formation in XX individuals, or a gain of function mutation in a gene downstream of SRY in the sex determining pathway.

Epizootic bovine abortion (EBA) is an arthropod-borne bacterial disease that causes significant economic loss for cattle producers in the western United States. The etiologic agent

A long intergenic non-coding RNA (lincRNA#1) is overexpressed in the horn bud region of polled (hornless) bovine fetuses, suggesting a potential role in horn bud suppression. Genome editing was used to test whether the absence of this sequence was associated with the horned phenotype. Two gRNAs with high mutation efficiencies targeting the 5′ and the 3′ regions flanking the lincRNA#1 sequence were co-injected with Cas9 as ribonucleoprotein complexes into bovine zygotes (n = 121) 6 h post insemination. Of the resulting blastocysts (n = 31), 84% had the expected 3.7 kb deletion; of these embryos with the 3.7 kb deletions, 88% were biallelic knockouts. Thirty-nine presumptive edited 7-day blastocysts were transferred to 13 synchronized recipient cows resulting in ten pregnancies, five with embryos heterozygous for the dominant PCPOLLED allele at the POLLED locus, and five with the recessive pp genotype. Eight (80%) of the resulting fetuses were biallelic lincRNA#1 knockouts, with the remaining two being mosaic. RT-qPCR analysis was used to confirm the absence of lincRNA#1 expression in knockout fetuses. Phenotypic and histological analysis of the genotypically (PCp) POLLED, lincRNA#1 knockout fetuses revealed similar morphology to non-edited, control polled fetuses, indicating the absence of lincRNA#1 alone does not result in a horned phenotype.

Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (P

Sperm mitochondrial function is essential for normal physiology and fertility, but the importance of mitochondrial activity to support specific sperm functions, such as motility, varies between species. It was previously believed that mitochondrial function was not necessary for bull sperm motility [1]; however, this theory is contradicted by recently reported findings that the upper fraction of bull sperm swim-up preparations had both high motility and elevated mitochondrial oxygen consumption rates [2]. The objective of this study was to investigate the relationship between mitochondrial function and motility in bull sperm. We hypothesized that sperm motility would be positively correlated with mitochondrial oxygen consumption (MITOX) but unaffected by pharmacological inhibition of electron transport chain (ETC) activity. This was accomplished by monitoring both mitochondrial oxygen consumption and motility parameters in the presence of mitochondrial effector drug treatments. Duplicate ejaculates were collected by electroejaculation from Black Angus bulls (n = 4). Oxygen consumption, as % air saturation (pO2; oxygen partial pressure), over time was monitored in the presence of 5 drug treatments: vehicle control, FCCP, Antimycin (ANTI), Oligomycin (Oligo), and FCCP + Oligomycin (FCCP + OLIGO). Duplicate aliquots were prepared for concurrent motility assessment by computer-assisted sperm analysis (CASA) at 6 and 30 min post-treatment (t6 and t30). The impact of treatments on pO2 (in % air saturation) over time were assessed by generalized linear mixed effects modeling (GLMM) which was also used to test for differences in average motility across treatment conditions and time points (t6 and t30). Pearson product-moment correlation was used to investigate relationships between oxygen consumption and motility parameters. Overall, pO2 differed over time between treatment conditions (p < 0.0001). When compared to the vehicle treatment, ANTI and OLIGO significantly inhibited oxygen consumption (p < 0.05, adjusted), and FCCP stimulated a marked increase in oxygen consumption. No significant differences in motility over time were observed between treatments, so comparison of motility parameters between treatment conditions was performed with pooled timepoints. Motility parameters were only observed to differ significantly from the vehicle with ANTI Treatment, for which significant decreases in numerous parameters, including total motility (p = 0.007), progressive motility (p = 0.01), DAP (p = 0.01), VAP (p = 0.01) and VSL (p = 0.02) were identified. For the vehicle treatment, correlational analysis did not reveal any significant correlations between pO2 and any motility parameters at t6; however, several significant correlations were identified at t30. Mean pO2 was negatively correlated with local motility (p < 0.01) and positively correlated with DCL, DAP, and VCL (p < 0.05). Results from this study suggest that bovine sperm motility is impacted by mitochondrial functionality, with ETC inhibition by ANTI causing significant reduction in motility parameters. This study also demonstrates the use of a new technology for the assessment of bovine sperm mitochondrial function. This modality for evaluation of bull sperm mitochondrial function will inform future efforts to understand bull sperm function and fertility and aid investigations into toxicological agents.

Background The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. Results By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. Conclusion The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

Risk assessments of genetically modified (GM) animals include evaluating whether milk and meat derived from the GM animal is as nutritious to humans and/or animals as traditionally-bred animals. The underlying assumption of this comparative approach is that traditionally-bred animals have a well-established history of safe use. The goal of this project was to provide empirical data on the development and nutritional composition of animal products derived from the offspring of a genome edited dairy bull, homozygous for the dominant PC Celtic POLLED allele. The bull was crossed with horned Hereford (HH) cows (pp) to obtain five male and one female heterozygous hornless (RC) calves. Hereford, Angus and Holstein bulls were also bred to Hereford cows to produce five HH calves, two polled Angus/Hereford (AH) calves, and three horned Holstein/Hereford (HO) calves (with 25% genetic identity to the hornless offspring), respectively as contemporary controls. Weights were recorded at 8 months, 1 year and slaughter (n = 9–16). Following calving, individual quarters of the RC heifer (RC.calf1), one HO heifer (HO.calf2), two horned HH heifers, and one contemporaneous hornless AH cow (3113) were milked at varying timepoints during the first seven weeks of lactation. Meat samples were collected from the 6 hornless RC calves and 3 horned HO comparator offspring after slaughter. Proximate analysis was conducted for meat and milk samples. Cattle that inherited a POLLED allele from the genome-edited bull showed no differences in their overall growth (Figure 1) or meat composition (Figure 2) when compared to contemporary controls. Milk composition varied between animals and by days in milk (DIM), although values were within the normal range reported in peer-reviewed literature (Figure 3). Bovine milk composition is known to be influenced by breed, nutrition, parity, and DIM, making it difficult to obtain appropriate comparators in experiments with a limited number of GM animals.

Although incidence of clinical hypocalcemia in postpartum dairy cows is low in US dairies, subclinical hypocalcemia after calving is common and has been associated with metabolic and infectious disease. It is widespread farm practice to feed a diet rich in anions to prepartum dairy cattle to support calcium homeostasis. However, this diet is typically discontinued at parturition, when calcium needs are still high. The objective of this trial was to determine the effects of extending metabolic acidification into the first 3 d of lactation in multiparous Holstein cows with the use of magnesium chloride (MgCl2) hexahydrate drenches on blood ionized calcium concentrations. Adult Holstein cows at a commercial dairy in their second or higher lactation, with a urine pH of 6.8 or less on the day of calving, were randomly assigned to either treatment or control groups, resulting in 13 cows in the treatment group and 14 cows in the control group. Treatment cows received 480 g of oral MgCl2 hexahydrate once daily for 3 d for continued acidification starting on the day of calving, whereas cows in the control group received no treatment. Urine pH was measured daily for 5 d, starting on the day of calving (0 DIM), to assess acidification status; blood was collected on day of calving (0 DIM), 2 DIM, and 4 DIM and analyzed for ionized calcium concentrations. Differences in blood ionized calcium and urine pH over time were compared using longitudinal data analysis. Urine pH was lower in treatment cows compared with control cows at 1, 2, and 3 DIM. Blood ionized calcium concentrations were different from baseline, taken at enrollment (0 DIM) and at 2 and 4 DIM in both treatment and control cows. However, no difference was detectable between treatment and control cows at 2 or 4 DIM with respect to blood ionized calcium concentrations. Oral supplementation with MgCl2 hexahydrate resulted in the desired acidification of urine pH in the treatment group, similar to feeding of an anionic close-up diet. Continued acidification of dairy cows until 2 DIM did not result in clinically meaningful higher blood calcium concentrations compared with controls, and further research is needed, to identify physiological reasons for this finding.

Genome editing offers an opportunity to introduce targeted gene insertions into livestock breeding programs. Molecular geneticists have typically employed a donor repair template and the homologous recombination (HR) pathway in somatic cells to introduce gene knock-ins into livestock genomes, followed by cloning. Editing embryos directly to achieve targeted gene knock-ins is inefficient, especially for introducing large DNA sequences. Here we report using a one-step method to produce a gene knock-in bull calf by cytoplasmic microinjection of CRISPR/Cas9 reagents into a bovine embryo. In vitro fertilized one-cell bovine zygotes were injected with a gRNA/Cas9 ribonucleoprotein complex and homology mediated end joining donor template containing the sex determining region Y (SRY) gene, the green fluorescent protein (gfp) reporter gene driven by the SV40 promoter, and one kilobase homology arms targeting the H11 safe harbor locus on bovine chromosome 17. Seven-day blastocysts were evaluated using fluorescent microscopy, and nine green fluorescent embryos were transferred to synchronized recipients. Ultrasound evaluation at 35 days revealed one pregnancy. In April 2020, a healthy 50 kg male calf was born. DNA was extracted from placenta, blood and a fibroblast line derived from the calf and analyzed for SRY-GFP knock-in, as well as genotypic sex. PCR and Sanger sequencing revealed the biallelic edit of the target location on chromosome 17, with the insertion of three or seven copies of the SRY-GFP construct in addition to donor plasmid backbone, or a 26 base pair insertion, and an XY genotype. Future analysis of the XX offspring inheriting the SRY gene on chromosome 17 from this knock-in bull will reveal whether inheritance of the bovine SRY gene is sufficient to trigger the male developmental pathway in cattle.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Epizootic bovine abortion (EBA) is an arthropod-borne bacterial disease that causes significant economic loss for cattle producers in the western United States. The etiologic agent, Pajaroellobacter abortibovis, is an intracellular pathogen that has yet to be cultivated in vitro, thereby requiring novel methodologies for vaccine development. A vaccine candidate, using live P. abortibovis-infected cells (P.a-LIC) harvested from mouse spleens, was tested in beef cattle. Over the course of two safety studies and four efficacy trials, safety risks were evaluated, and dosage and potencies refined. No incidence of anaphylaxis, recognized health issues or significant impact upon conception rates were noted. Vaccination did result in subclinical skin reactions. Early fetal losses were noted in two trials and were significant when the vaccine was administered within 21 days prior to conception. Administration of the EBA agent (EBAA) vaccine as a single dose, at a potency of 500 P.a–LIC, 56 days prior to breeding, provided 100% protection with no early fetal losses. Seroconversion occurred in all animals following EBAA vaccination and corresponded well with protection of the fetus from epizootic bovine abortion.

Optimal mineral nutrition is required for cattle reproduction, immune function, and structural development. Formal evaluation of the current mineral status of California beef cattle is currently lacking. In 2017, a survey was initiated that evaluated a panel of 10 different minerals in 14 counties across California. Samples were collected from 555 cattle at 50 different ranches. Region of the state significantly affected herd mineral status. Herd use of supplements was also significant, and increased most blood levels of the mineral(s) targeted for supplementation. Forage source was idiosyncratic on its effect of mineral status. Previous blood survey data showed selenium to be widely deficient in California cattle in the 1970s and 1980s, but in this case, it was generally adequate in all areas of California. This indicates a good producer understanding of where supplementation is needed. Copper deficiency was more widespread in the southern region when compared with further north. Zinc deficiency was seen ubiquitously statewide, with 36% of animals being deficient. Manganese has been largely ignored in California. This study is the first known documentation of manganese levels in the state. Sampling found 92% of cattle fell below critical manganese levels. However, further research to better define manganese critical levels is probably warranted. The status of other minerals is presented.

The unattainability of the absolute zero of temperature is proved by using Carnot’s theorem. Hence this unattainability is distinct from the Planck-Fer-mi statement of the Third Law of Thermodynamics that the entropy vanishes at T=0. It is shown that the isothermal compressibility KT is in general larger than the adiabatic compressibility Ks and the difference KT − Ks vanishes in the low temperature limit.

CASE DESCRIPTION A 2-year-old primiparous miniature Scottish Highland cow with an unknown breeding date was evaluated for suspected hydrops. CLINICAL FINDINGS Transabdominal and transrectal ultrasonographic examination identified a large amount of hypoechoic fluid within an enlarged uterus; the fetus could not be identified. Presence of a severely distended uterus and concerns regarding associated health risks to the cow led to the decision to induce labor. Although fluids were expelled, parturition did not progress further over the following 48 hours. Vaginal examination revealed a partially dilated cervix and an abnormally shaped fetus that was too large to pass vaginally. TREATMENT AND OUTCOME Supportive care was provided to the cow, and a stillborn bull calf was delivered by cesarean section. Grossly evident chondrodystrophic dwarfism with hydrocephalus, compatible with so-called bulldog calf malformations, was confirmed by diagnostic imaging and histopathologic evaluation. The cow recovered from surgery uneventfully and was discharged from the hospital the following day. Genetic analysis of DNA from hair roots collected from the sire and dam confirmed both were carriers of an aggrecan-1 gene mutation (bulldog dwarfism1) previously associated with dwarfism and bulldog calf malformations in Dexter cattle. CLINICAL RELEVANCE To our knowledge, this is the first reported case of bulldog calf malformations associated with an aggrecan-1 gene mutation in miniature Scottish Highland cattle, confirming that at least 1 genetic mutation associated with this condition is found in cattle breeds other than Dexter. The findings highlighted the clinical importance of testing for known genetic diseases in breeding cattle, particularly among miniature breeds.

One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.

To compare the use of controlled intravaginal drug release (CIDR, EAZI-BREED™ CIDR® Sheep, USA, Zoetis) with the ram effect, a combination of CIDR and ram effect and the addition of P.G.600® to each of these scenarios on their contribution to inducing the onset of cyclicity and their ability to improve reproductive efficiency in a commercial sheep flock, a randomized clinical trial was conducted. Multiparous ewes (n = 283) were randomly assigned to one of six treatments prior to intact male introduction on day 0: only CIDR devices (day −5 to day 0) (n = 40), exposure to two vasectomized rams (d-14 to d0) (n = 55), CIDR (d-5 to d0) and an intramuscular injection of P.G.600® (d0) (n = 40), exposure to two vasectomized rams (d-14 to d0) and treated with CIDR devices (day −5 to day 0) (n = 40). exposure to vasectomized rams (d-14 to d0), CIDR (d-5 to d0) and an intramuscular injection ofP.G.600®, (d0) (n = 38), and a control group, without exposure to vasectomized rams, CIDRs or P.G.600® (n = 70). Ewes were exposed to intact males on day 0. Days to lambing from the earliest expected lambing date at 150 days post introduction of rams was compared between groups with survival analysis. The mean survival time was significantly shorter in all groups that used CIDR compared to the control group (P

Reflection symmetry properties play important roles for the stability of crystal lattices in which electrons and phonons move. Based on the reflection symmetry properties, cubic, tetragonal, orthorhombic, rhombohedral (trigonal) and hexagonal crystal systems are shown to have three-dimensional (3D) k-spaces for the conduction electrons (“electrons”, “holes”). The basic stability condition for a general crystal is the availability of parallel material planes. The monoclinic crystal has a 1D k-space. The triclinic has no k-vectors for electrons, whence it is a true insulator. The monoclinic (triclinic) crystal has one (three) disjoint sets of 1D phonons, which stabilizes the lattice. Phonons’ motion is highly directional; no spherical phonon distributions are generated for monoclinic and triclinic crystal systems.

Following Ashcroft and Mermin, the conduction electrons (“electrons” or “holes”) are assumed to move as wave packets. Dirac’s theorem states that the quantum wave packets representing massive particles always move, following the classical mechanical laws of motion. It is shown here that the conduction electron in an orthorhombic crystal moves classical mechanically if the primitive rectangular-box unit cell is chosen as the wave packet, the condition requiring that the particle density is constant within the cell. All crystal systems except the triclinic system have k-vectors and energy bands. Materials are conducting if the Fermi energy falls on the energy bands. Energy bands and gaps are calculated by using the Kronig-Penny model and its 3D extension. The metal-insulator transition in VO2 is a transition between conductors having three-dimensional and one-dimensional k-vectors.

One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1−/− fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

Genome editing using programmable nucleases has revolutionized biomedical research. CRISPR-Cas9 mediated zygote genome editing enables high efficient production of knockout animals suitable for studying development and relevant human diseases. Here we report efficient disabling pancreatogenesis in pig embryos via zygotic co-delivery of Cas9 mRNA and dual sgRNAs targeting the PDX1 gene, which when combined with chimeric-competent human pluriopotent stem cells may serve as a suitable platform for the xeno-generation of human tissues and organs in pigs.

Based on the idea that different temperatures generate different conduction electron densities and the resulting carrier diffusion generates the thermal electromotive force (emf), a new formula for the Seebeck coefficient (thermopower) S is obtained: S=(2/3)ln2(qn)-1eFkBD0, where kB is the Boltzmann constant, and q, n, eF, D0 are charge, carrier density, Fermi energy, density of states at eF, respectively. Ohmic and Seebeck currents are fundamentally different in nature, and hence, cause significantly different behaviors. For example, the Seebeck coefficient S in copper (Cu) is positive, while the Hall coefficient is negative. In general, the Einstein relation between the conductivity and the diffusion coefficient does not hold for a multicarrier metal. Multi-walled carbon nanotubes are superconductors. The Seebeck coefficient S is shown to be proportional to the temperature T above the superconducting temperature Tc based on the model of Cooper pairs as carriers. The S follows a temperature behavior, , where Tg ’= constant, at the lowest temperatures.

A compact and fast electromagnetic calorimeter prototype was designed, built, and tested in preparation for a next-generation, high-rate muon ( g − 2 ) experiment. It uses a simple assembly procedure: alternating layers of 0.5-mm-thick tungsten plates and 0.5-mm-diameter plastic scintillating fiber ribbons. This geometry leads to a detector having a calculated radiation length of 0.69 cm, a Moliere radius of 1.73 cm, and a measured intrinsic sampling resolution term of ( 11.8 ± 1.1 ) % / E ( GeV ) , in the range 1.5–3.5 GeV. The construction procedure, test beam results, and GEANT-4 comparative simulations are described.

Behavioral preadaptations can provide an accommodating environment in which novel morphological characters may be selected. A very recent morphological mutation, flatwing, has caused the loss of male song in field crickets (Teleogryllus oceanicus) from the Hawaiian island of Kauai. Previous studies in this and related species have shown that females require male courtship song to mount males, but flatwing T. oceanicus males still achieve matings. Females from Kauai may have a relaxed requirement for male courtship song, or flatwing males may compensate for their inability to sing by altering other courtship behaviors. We tested whether male courtship and female responses to male courtship were preadapted in a way that facilitated the spread of the male wing mutation or if parallel changes in male courtship and female responses accompanied the mutation. We performed mating trials in 2 captive-bred populations to assess how mating behavior varied depending on the presence or absence of courtship song playback. The first was an ancestral population from Kauai established prior to the emergence of the flatwing mutation, and the second was derived from Kauai after the mutation became prevalent. Mating behaviors did not differ qualitatively or quantitatively between the ancestral and current populations, and females from both accepted males for mating in the absence of courtship song. Our results provide direct evidence that a mechanism allowing flatwing males to mate with females was in place before the mutation actually arose on Kauai and demonstrate how preexisting behavior facilitated the rapid spread of a novel morphological mutation. Copyright 2008, Oxford University Press.

In the muon ( g - 2 ) experiment at Brookhaven National Laboratory, the spin precession frequency ω a is obtained from a standard χ 2 minimization fit applied to the time distribution of decay electrons. The unusually high accuracy ( ∼ 0.5 ppm ) of the experiment puts stringent requirements on the quality of the fit and the level of understanding of the statistical properties of the fitted parameters. We discuss the properties of the fits and their implications on the derived value for ω a , including estimates of the effect of an imperfect fit function, methods of including additional external information to reduce the error, the effects of splitting the data into many smaller subsets of data, applying different weighting methods to the data using energy information, and various tests of data suitability.

The experimental method together with the analysis method and results of the data taken in 2000 and prospects of the muon anomalous magnetic and electric dipole moment experiments are presented here., 12 pages, 20 figures. Proceedings of my plenary talk at ICHEP02, Amsterdam, 31 July 2002

The status of the muon g — 2 experiment at the AGS facility of Brookhaven National Laboratory is discussed. Data obtained in 1999 with positive muons has been analyzed and published. The final data set contained 0.95 × 10 9 events and had an accuracy of 1.3 ppm . Approximately four times more data with positive muons and three times more data with negative muons were obtained in 2000 and 2001, respectively. These data were obtained with a more uniform magnetic field and with different storage ring tunes. An accuracy of the order of 0.5 ppm is anticipated.

The measurement of the (g−2) value of leptons provides a unique test of theory since it is the only quantity (unlike charge and mass) calculable in the framework of the Standard Model of elementary particles. The muon (g−2) experiment E821 is currently in progress at Brookhaven National Laboratory. Four data taking runs for positive muons and one run for negative muons were successfully accomplished in 1997–2000 and 2001, respectively. Results of the 1997–2000 runs have been published, thus completing our experiment for μ+. Data analysis for the 2001 run for μ− is currently in progress. To provide measurement of \(a_\mu = \tfrac{1} {2}(g - 2)_{\mu ^ - }\) at the same level of accuracy as for \(a_{\mu ^ + } = \tfrac{1} {2}(g - 2)_{\mu ^ + }\), we need to have one more data taking run.

A precise measurement of the anomalous g value, a(mu)(+) = (g - 2)/2, for the positive muon has been made at the Brookhaven Alternating Gradient Synchrotron. The result a(mu)(+) = 11 659 202(14)(6) x 10(-10) (1.3 ppm) is in good agreement with previous measurements and has an error one third that of the combined previous data. The current theoretical value from the standard model is a(mu) (SM)= 11 659 159.6(6.7) x 10(-10) (0.57 ppm) and a(mu)(exp)-a(mu)(SM) = 42(16) x 10(-10) in which a(mu)(exp) is the world average experimental value.

By the end of an excellent data taking in 1999, we collected approximate to1 billion decay positrons with energy greater than 2 GeV and 30 mus after injection. The analysis of the 1999 data set were performed in parallel by various teams in the collaboration and each team provides a different approach to the analysis. The projected errors are expected to be of order 1.3 ppm statistical and below 0.5 ppm systematic. The data obtained in the 2000 run contains,approximate to4 times more decay positrons compared to 1999.

Tandem instruments, such as triple-quadrupole or quadrupole-time-of-flight mass spectrometers, often use collisional damping ion guides for the purpose of cooling and focusing the primary ion beam, or as a collision cell for tandem mass spectrometry experiments. A small axial field to reduce the ion residence time in such devices can give considerable improvements in performance. Reduction of the residence time reduces adduct formation caused by unwanted gas-phase reactions and allows multiple reaction monitoring (MRM) and some other types of scan to be carried out more rapidly without the risk of reaction cross-talk. Here we propose a novel electrode arrangement for creation of a suitable axial field in a multipole ion guide without significantly reducing the m/z window. As an example, a second set of four electrodes is added to an existing quadrupole collision cell. The same DC potential is applied to all four extra electrodes, which are shaped in the longitudinal direction to create a suitable axial field inside the device. The potential of these electrodes and the DC bias of the main rods determine the axial field strength. The idea was tested on a modified quadrupole collision cell of a MALDI-QqTOF instrument and experimental results are presented here, as well as a simplified theory of operation.

The muon (g-2) experiment at Brookhaven completed a first run in June and July 1997. The main components of the experiment, which include the superconducting inflector, the superferric storage ring, the electrostatic quadrupoles and the lead-scintillating fiber electron calorimeters, have been commissioned satisfactorily. Our first measurement of the ratio R of the spin precession frequency of the positive muon relative to that of a free proton, R=(3.707219/spl plusmn/0.000048)/spl times/10/sup -3/, is in good agreement with the previous CERN measurements for /spl mu//sup +/ and /spl mu//sup -/, and has approximately the same uncertainty as each of these measurements. In spring 1998 a muon kicker was installed and successfully tested in the storage ring magnet and a significant improvement in the knowledge of the muon g-factor is expected from upcoming runs in August 1998 and January 1999.

The muon g-2 experiment at Brookhaven has successfully completed two exploratory runs using pion injection and direct muon injection for checkout and initial data taking. The main components of the experiment, which include the pion beam line, the superconducting storage ring and inflector magnets, the muon kicker and the lead-scintillating fiber calorimeters have been satisfactorily commissioned. First results on the anomalous magnetic moment of the positive muon from pion injection are in good agreement with previous experimental results for a(mu+) and a(mu-) from CERN and of comparable accuracy (13 ppm). Analysis of the 1998 muon injection run is in progress and expected to improve the precision to about 4 ppm. A first production run is scheduled for January 1999 with the goal of reaching the 1 ppm error level.

We present a detailed report of the method, setup, analysis and results of a precision measurement of the positive muon lifetime. The experiment was conducted at the Paul Scherrer Institute using a time-structured, nearly 100%-polarized, surface muon beam and a segmented, fast-timing, plastic scintillator array. The measurement employed two target arrangements; a magnetized ferromagnetic target with a ~4 kG internal magnetic field and a crystal quartz target in a 130 G external magnetic field. Approximately 1.6 x 10^{12} positrons were accumulated and together the data yield a muon lifetime of tau_{mu}(MuLan) = 2196980.3(2.2) ps (1.0 ppm), thirty times more precise than previous generations of lifetime experiments. The lifetime measurement yields the most accurate value of the Fermi constant G_F (MuLan) = 1.1663787(6) x 10^{-5} GeV^{-2} (0.5 ppm). It also enables new precision studies of weak interactions via lifetime measurements of muonic atoms., Comment: Submitted for publication in Phys. Rev. D (32 pages, 28 figures, 5 tables)

The aim of this study was to determine the incidence of echocardiographically detectable patent foramen ovale (PFO) in elderly patients who have strokes from cerebral infarction, as well as to assess the association between PFOs and other risk factors for stroke disease. Forty-three patients agedor = 65 years admitted consecutively with cerebral infarction shown on computerised tomography of the brain were assessed using transoesophageal echocardiography. A PFO was present in 8 (19%) of the 43 patients. Four patients (50%) in this PFO group had stroke risk factors. Two were in atrial fibrillation and, of the six patients in sinus rhythm, a risk factor was present in two, both of whom had diabetes mellitus. A PFO was not detected in 35 patients. Twenty-nine (83%) of the patients in this group had risk factors, with 18 having two or more. Seven patients were in atrial fibrillation. Of the 28 patients in sinus rhythm, risk factors were present in 22 (78%).

We report a measurement of the positive muon lifetime to a precision of 1.0 parts per million (ppm); it is the most precise particle lifetime ever measured. The experiment used a time-structured, low-energy muon beam and a segmented plastic scintillator array to record more than 2 x 10^{12} decays. Two different stopping target configurations were employed in independent data-taking periods. The combined results give tau_{mu^+}(MuLan) = 2196980.3(2.2) ps, more than 15 times as precise as any previous experiment. The muon lifetime gives the most precise value for the Fermi constant: G_F(MuLan) = 1.1663788 (7) x 10^-5 GeV^-2 (0.6 ppm). It is also used to extract the mu^-p singlet capture rate, which determines the proton's weak induced pseudoscalar coupling g_P., Accepted for publication in Phys. Rev. Lett

Three independent searches for an electric dipole moment (EDM) of the positive and negative muons have been performed, using spin precession data from the muon g-2 storage ring at Brookhaven National Laboratory. Details on the experimental apparatus and the three analyses are presented. Since the individual results on the positive and negative muon, as well as the combined result, d=-0.1(0.9)E-19 e-cm, are all consistent with zero, we set a new muon EDM limit, |d| < 1.9E-19 e-cm (95% C.L.). This represents a factor of 5 improvement over the previous best limit on the muon EDM., 19 pages, 15 figures, 7 tables