scholarly journals Using Lentivirus and CRISPR to Modify Cattle Embryonic Genes

2021 ◽  
Author(s):  
◽  
Jevon Upton
Keyword(s):  
Genetic Modification ◽  
Zona Pellucida ◽  
Environmental Changes ◽  
Early Stage ◽  
High Titer ◽  
Genetic Material ◽  
Ease Of Use ◽  
Disease Genes ◽  
Protective Agent ◽  
Bovine Embryos

<p>Developing transgenic livestock has become popular in recent years after advances in the field of genetic editing. Cattle are one of the main exports in New Zealand and are a prime target for new genetic editing tools. Applications of genetic editing in cattle can extend to increases in production, and elimination of disease genes. Due to its ease of use, CRISPR/Cas9 has become one of the most popular methods of editing genes, hence this was employed in the research. Cattle embryos in culture are very sensitive to environmental changes and for this reason, a delivery vector is necessary to deliver the genetic material as traditional transfection methods cause high rates of embryo death. The zona pellucida, a glycoprotein coat surrounding the embryo, acts as a protective agent against viral vectors, and needed to be considered in the research. This research aimed to create a novel, high titer lentivirus particle capable of transducing bovine embryos, and causing subsequent genetic modification by integration of CRISPR/Cas9 into the genome. Using fluorescent reporters, viral transduction was monitored. The research found that after optimizing transfection protocols, high-titer lentiviral particles can be produced and can infect bovine embryos. Zona pellucida removal experiments revealed over-digestion in early stage embryos, however, this was not observed in compact morulas. Removing the zona allowed for successful transduction of bovine embryos, resulting in transgenic cells expressing eGFP. While CRISPR/Cas9 experiments were in preliminary stages, these indicated eGFP knock-out in certain eGFP-HEK293T cells. Though challenges were encountered throughout the research process, solutions were explored, and it was shown that transgenic bovine embryos using lentiviral gene delivery can be produced. This indicates the high likelihood that CRISPR/Cas9 systems can be delivered this way, inducing targeted genetic modification.</p>

scholarly journals Using Lentivirus and CRISPR to Modify Cattle Embryonic Genes

2021 ◽  
Author(s):  
◽  
Jevon Upton
Keyword(s):  
Genetic Modification ◽  
Zona Pellucida ◽  
Environmental Changes ◽  
Early Stage ◽  
High Titer ◽  
Genetic Material ◽  
Ease Of Use ◽  
Disease Genes ◽  
Protective Agent ◽  
Bovine Embryos

<p>Developing transgenic livestock has become popular in recent years after advances in the field of genetic editing. Cattle are one of the main exports in New Zealand and are a prime target for new genetic editing tools. Applications of genetic editing in cattle can extend to increases in production, and elimination of disease genes. Due to its ease of use, CRISPR/Cas9 has become one of the most popular methods of editing genes, hence this was employed in the research. Cattle embryos in culture are very sensitive to environmental changes and for this reason, a delivery vector is necessary to deliver the genetic material as traditional transfection methods cause high rates of embryo death. The zona pellucida, a glycoprotein coat surrounding the embryo, acts as a protective agent against viral vectors, and needed to be considered in the research. This research aimed to create a novel, high titer lentivirus particle capable of transducing bovine embryos, and causing subsequent genetic modification by integration of CRISPR/Cas9 into the genome. Using fluorescent reporters, viral transduction was monitored. The research found that after optimizing transfection protocols, high-titer lentiviral particles can be produced and can infect bovine embryos. Zona pellucida removal experiments revealed over-digestion in early stage embryos, however, this was not observed in compact morulas. Removing the zona allowed for successful transduction of bovine embryos, resulting in transgenic cells expressing eGFP. While CRISPR/Cas9 experiments were in preliminary stages, these indicated eGFP knock-out in certain eGFP-HEK293T cells. Though challenges were encountered throughout the research process, solutions were explored, and it was shown that transgenic bovine embryos using lentiviral gene delivery can be produced. This indicates the high likelihood that CRISPR/Cas9 systems can be delivered this way, inducing targeted genetic modification.</p>

scholarly journals STUDIES ON THE IMMUNOTHERAPY OF RUNT DISEASE IN RATS

1969 ◽  
Vol 129 (4) ◽  
pp. 647-661 ◽  
Author(s):  
Willys K. Silvers ◽  
R. E. Billingham
Keyword(s):  
Early Stage ◽  
Fatal Outcome ◽  
Dosage Level ◽  
Cell Suspensions ◽  
Necessary Condition ◽  
Target Cells ◽  
Protective Agent ◽  
Cutaneous Manifestations ◽  
Therapeutic Value ◽  
Tissue Antigens

Using rats of the Lewis and BN (Ag-B locus incompatible) isogenic strains, a comparative study has been made of the capacity to prevent or mitigate the development of runt disease with: (a) lymph node cell suspensions from normal adult BN rats, (b) node cells, or (c) serum from donors sensitized against Lewis tissue antigens, or (d) heterologous anti-lymphocyte serum (ALS) raised in rabbits against rat thymocytes. Following a standard intravenous or intraperitoneal inoculation of 20 x 106 Lewis node cells into neonatal BN hosts, there are cutaneous manifestations of runt disease within 11–15 days and death invariably takes place within 20 days. However, complete protection is afforded by administration of a similar number of normal BN node cells via a different vein, or admixed with the otherwise harmful Lewis node cells. However, timing of the administration was crucially important—precedence or delay by as little as 4 hr resulted in a great impairment of protection. When the inoculations of the two cell suspensions were separated by 24 hr, no protection was afforded. These and other observations suggested that a necessary condition for protection of the hosts by unsensitized isologous cells requires that they establish a prompt and intimate confrontation with the homologous target cells. At the same dosage level, suspensions of node cells from sensitized isologous donors were much more effective therapeutically, saving the lives of 92% of treated subjects when administered after a delay of 3 days, and of 19% when the delay was 4 or 5 days. Of the various immunotherapeutic agents studied, daily injections of 0.2 ml of isoantiserum gave the best results, and could totally reverse the course of the disease even when initiated at age 10–13 days and subjects already presented symptoms. ALS, although inferior to isoantiserum at the dosage levels tested, proved to be superior to sensitized isologous cells as a protective agent, since the initiation of daily injections after delays of 6 or 8 days were still effective. The observations that delayed treatments of infant rats with isoantisera or ALS resulted in complete recoveries sustain the thesis that the lesions responsible for the fatal outcome of runt diseases are not inflicted at a very early stage. The efficacy of both isoantisera and ALS as a means of inhibiting the progression of homologous disease also suggests that they may have therapeutic value in situations where this condition is encountered.

Hybridization capture of Larix candidate adaptive genes from sedimentary ancient DNA.

2021 ◽  
Author(s):  
Stefano Meucci ◽  
Luise Schulte ◽  
Kathleen R. Stoof-Leichsenring ◽  
Stefan Kruse ◽  
Konstantin Krutovsky ◽  
...  
Keyword(s):  
Ancient Dna ◽  
Dna Sequences ◽  
Environmental Changes ◽  
Genetic Material ◽  
Siberian Larch ◽  
Shotgun Sequencing ◽  
Adaptive Genetic Variation ◽  
Hybridization Capture ◽  
Sequencing Method ◽  
Larch Species

&lt;p&gt;Siberian larch forests dominate large areas of northern Russia and contribute important roles for the world&amp;#180;s ecosystem. In order to understand the past dynamics of larches and their adaptive genetic variation, sedimentary ancient DNA (sedaDNA) extracted from lake sediment cores is a crucial source of genetic material. The difficulty of retrieving extremely rare DNA sequences from samples reaching back up to 25000 years in age, is challenging. Previous studies (Schulte et al.) showed that the hybridization capture allowed an enrichment of targeted sequences by several orders of magnitude in comparison to shotgun sequencing method. Therefore, we established for the first time, a hybridization capture method targeting 65 candidate adaptive genes laying on the Larix nuclear genome. Our preliminary results showed the ability of our newly established method to enrich extremely rare DNA sequences of the targeted Larix candidate adaptive genes, which were not retrieved by shotgun sequencing method applied on the same samples. Furthermore, the results allowed to detect and compare specific nucleotide polymorphism of adaptive candidate genes among sedaDNA samples distributed in space and time. The establishment of this new method is laying the basis to investigate possible adaptive variation of larch species acquired across the dry and cold conditions of the Last Glacial Maximum (LGM); as well as their possible advantages or disadvantages in relation to the current environmental changes toward dry and warm conditions.&lt;/p&gt;

scholarly journals Sea-ice edge is more important than closer open water access for foraging Adélie penguins: evidence from two colonies

2020 ◽  
Vol 640 ◽  
pp. 215-230
Author(s):  
C Michelot ◽  
A Kato ◽  
T Raclot ◽  
K Shiomi ◽  
P Goulet ◽  
...  
Keyword(s):  
Sea Ice ◽  
Environmental Changes ◽  
Early Stage ◽  
Open Water ◽  
Gps Tracking ◽  
Ice Edge ◽  
Sea Ice Edge ◽  
Study Sites ◽  
The Impact ◽  
Adélie Penguins

Sentinel species, like Adélie penguins, have been used to assess the impact of environmental changes, and their link with sea ice has received considerable attention. Here, we tested if foraging Adélie penguins from 2 colonies in East Antarctica target the distant sea-ice edge or take advantage of closer open waters that are readily available near their colony. We examined the foraging behaviour of penguins during the incubation trips of females in 2016 and males in 2017, using GPS tracking and diet data in view of daily sea-ice data and bathymetry. In 2016-2017, sea-ice cover was extensive during females’ trips but flaw leads and polynyas were close to both study sites. Sea ice receded rapidly during males’ trips in 2017-2018. Despite close open water near both colonies in both years, females and males preferentially targeted the continental slope and the sea-ice edge to forage. In addition, there was no difference in the diet of penguins from both colonies: all penguins fed mostly on Antarctic krill and males also foraged on Antarctic silverfish. Our results highlight the importance of the sea-ice edge for penguins, an area where food abundance is predictable. It is likely that resource availability was not sufficient in closer open water areas at such an early stage in the breeding season. The behaviours displayed by the penguins from both colonies were similar, suggesting a common behaviour across colonies in Terre Adélie, although additional sites would be necessary to confirm this hypothesis.

scholarly journals NMR microsystem for label-free characterization of 3D nanoliter microtissues

2020 ◽  
Author(s):  
Marco Grisi ◽  
Gaurasundar M. Conley ◽  
Kyle J. Rodriguez ◽  
Erika Riva ◽  
Lukas Egli ◽  
...  
Keyword(s):  
Early Stage ◽  
Cmos Technology ◽  
Time Frame ◽  
Ease Of Use ◽  
Chemical Information ◽  
Spectroscopic Techniques ◽  
Label Free ◽  
Alcoholic Fatty Liver ◽  
Non Invasive

AbstractPerforming chemical analysis at the nanoliter (nL) scale is of paramount importance for medicine, drug development, toxicology, and research. Despite the numerous methodologies available, a tool for obtaining chemical information non-invasively is still missing at this scale. Observer effects, sample destruction and complex preparatory procedures remain a necessary compromise1. Among non-invasive spectroscopic techniques, one able to provide holistic and highly resolved chemical information in-vivo is nuclear magnetic resonance (NMR)2,3. For its renowned informative power and ability to foster discoveries and life-saving applications4,5, efficient NMR at microscopic scales is highly sought after6–10, but so far technical limitations could not match the stringent necessities of microbiology, such as biocompatible handling, ease of use, and high throughput. Here we introduce a novel microsystem, which combines CMOS technology with 3D microfabrication, enabling nL NMR as a platform tool for non-invasive spectroscopy of organoids, 3D cell cultures, and early stage embryos. In this study we show its application to microlivers models simulating non-alcoholic fatty liver disease (NAFLD), demonstrating detection of lipid metabolism dynamics in a time frame of 14 days based on 117 measurements of single 3D human liver microtissues.

The Spermatozoon of Actinia Equina L. Var. Mesembryanthemum

1980 ◽  
Vol 60 (1) ◽  
pp. 193-204 ◽  
Author(s):  
A. U. Larkman ◽  
M. A. Carter
Keyword(s):  
Fine Structure ◽  
Sexual Reproduction ◽  
Recent Work ◽  
Developmental Stages ◽  
Early Stage ◽  
Genetic Material ◽  
Sea Anemones ◽  
Free Living ◽  
Pass Through ◽  
Parent Female

Actinia equina var. mesembryanthemum, the beadlet anemone (Stephenson, 1935), is a very common and widely distributed littoral anthozoan, whose sexual reproduction shows several interesting characteristics. Adult sea anemones of both sexes brood planulae and more advanced developmental stages within the gastrovascular cavity, although earlier embryonic stages are rarely found brooded in this way. Chia & Rostron (1970) suggest that embryos are expelled from the parent female anemone at an early stage and pass through a free-living phase before re-entering anemones of either sex for brooding. However, recent work (Cain, 1974) suggests that juvenile anemones are genetically related to the adult anemones in which they are brooded, and also the distribution of genetic material during sexual reproduction appears to be abnormal (Carter & Thorp, 1979). In an attempt to achieve a better understanding of the unusual sexual reproduction of this species, an ultrastructural investigation of gametogenesis was undertaken. This paper describes the fine structure of the spermatozoon within the testis.

205 INCREASED PREGNANCY RATES OF POOR QUALITY BOVINE EMBRYOS BY ASSISTED HATCHING

2007 ◽  
Vol 19 (1) ◽  
pp. 219
Author(s):  
A. Taniyama ◽  
Y. Watanabe ◽  
Y. Nisino ◽  
T. Inoue
Keyword(s):  
Embryo Transfer ◽  
Zona Pellucida ◽  
Poor Quality ◽  
Pregnancy Rates ◽  
Assisted Hatching ◽  
Bovine Embryos ◽  
Production Rates ◽  
Chi Square ◽  
Chi Square Test ◽  
Rectal Palpation

Embryo transfer after superovulation is commonly used for efficient embryo and animal production and for genetic improvement in cattle. However, the quality of collected embryos varies greatly, which affects pregnancy rate. Usually, poor quality embryos are related to low pregnancy rates after embryo transfer and low viability after cryopreservation. Therefore, it is important to improve chances for survival of poor quality embryos after embryo transfer. The objective of this experiment was to improve pregnancy rates by applying the assisted hatching technique to poor quality embryos. Embryos were collected from Japanese Black cows after superovulation on Day 7 post-insemination. After being washed, embryos were morphologically classified. Embryos having more than 30% degenerated cells were assigned as poor quality embryos. The assisted hatching of embryos (cutting the zona pellucida) was performed under a stereoscope or an inverted microscope by making a cutting slit on the zona pellucida for about 20% of its circumference using a micromanipulator equipped with a cutting needle and holding pipette. After cutting, single or two embryos were transferred fresh to one uterine horn of recipient cows on Day 7 of the estrous cycle. Pregnancy and calf production rates were compared between 2 embryo transfer groups composed of fresh zona-cut embryos (ZC group) or fresh embryos with non-cut zonae pellucidae (NZC group). Pregnancy rates were determined by rectal palpation on Day 45, and calf production rates were calculated by the following formula: number of calves born/number of pregnancies. Statistical analysis was carried out using the chi-square test. Pregnancy rates of poor quality embryos in the double ET ZC group (60.3%; 44 pregnancies/73 transfers) were significantly higher (P &lt; 0.05) than those in the single ET NZC group (25.0%; 6 pregnancies/24 transfers) and in the single ET ZC group (44.0%; 37 pregnancies/84 transfers). Calf production rates were 67.3%, 45.5%, and 35.6% for the double ET ZC group, the double ET NZC group, and the single ET ZC group, respectively. Pregnancy rates of poor quality bovine embryos after double ET were remarkably improved by assisted hatching compared with those of single ET with non-assisted hatching. These results suggest that the combined methods of assisted hatching and double ET may be beneficial to produce calves from poor quality embryos.

185 EMBRYONIC LOSS IN PIGS ASSOCIATED WITH OVIDUCT TRANSPLANTATION OF EARLY-STAGE EMBRYOS WITH DAMAGES IN THE ZONA PELLUCIDA

2006 ◽  
Vol 18 (2) ◽  
pp. 200
Author(s):  
S. Ueno ◽  
M. Kurome ◽  
R. Tomii ◽  
K. Hiruma ◽  
N. Maeda ◽  
...  
Keyword(s):  
Immune Response ◽  
Zona Pellucida ◽  
Nuclear Transfer ◽  
Early Stage ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Embryo Recovery ◽  
Embryonic Loss ◽  
The Impact

It is assumed that if porcine early-stage embryos with damages in their zonae pellucidae are transplanted to the recipient's oviduct, they may suffer from mechanical and immunological stresses by oviduct contraction and the recipient's immune response. This study aimed to examine the impact of zona pellucida damages, which might arise during nuclear transfer and intra cytoplasmic sperm injection (ICSI), on the development and survival of transplanted embryos. Cumulus-oocyte complexes were collected from ovaries obtained at a local slaughterhouse and matured in vitro in NCSU23 to prepare MII-stage oocytes. The zonae pellucidae of these oocytes were either penetrated with 8- to 10-�m square-ended microinjection pipettes or incised with 35- to 40-�m beveled enucleation pipettes. Intact oocytes were used as controls. The oocytes were electroactivated to induce parthenogenesis and transplanted to the oviducts of estrus-synchronized recipient gilts (estrus-synchronized with 1000 IU eCG and 1500 IU hCG). After 5 to 7 days, the recipient uteri were flushed with PBS supplemented with 1% fetal bovine serum (FBS) to collect embryos, and their development (morula-blastocyst stage embryos/collected embryos) and survival (viable embryos/collected embryos) were determined. In total, 221 zona-penetrated, 129 zona-incised, and 57 intact embryos were transplanted to four, two and two gilts, respectively. The efficiency of embryo recovery was similar in all groups (59.0 to 81.8%). However, the zona-penetrated and zona-incised embryos showed inconsistent development and survival compared with controls; the development and survival rate were 92.6% (25/27) to 96.7% (29/30) and 77.8% (21/27) to 96.7% (29/30) in control embryos, respectively, whereas those of zona-penetrated embryos were 57.1% (28/49) to 95.7% (22/23) and 8.2% (4/49) to 78.3% (18/30), and those of zona-incised embryos were 47.6% (30/63) to 92.3% (36/39) and 23.8% (15/63) to 92.3% (22/23), respectively. Large foci of cells that appeared to be macrophage giant cells were observed at the surface or inside of the degenerated zona-damaged embryos. These results indicate that the recipient's immune response may impair development after transplantation of the embryo to the oviduct, when there is damage in the zona pellucida. This may be one of the factors attributable to the reduced efficiency of live progeny production by ICSI and nuclear transfer. This work was supported by PROBRAIN.

154 THE QUANTITY OF BOVINE VIRAL DIARRHEA VIRUS ASSOCIATED WITH SINGLE ZONA PELLUCIDA-INTACT IN VITRO-PRODUCED BOVINE EMBRYOS FOLLOWING ARTIFICIAL EXPOSURE

2008 ◽  
Vol 20 (1) ◽  
pp. 157
Author(s):  
J. A. Gard ◽  
M. D. Givens ◽  
P. K. Galik ◽  
K. P. Riddell ◽  
M. S. D. Marley ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Bovine Viral Diarrhea Virus ◽  
Zona Pellucida ◽  
Quantitative Polymerase Chain Reaction ◽  
Bovine Viral Diarrhea ◽  
Bovine Embryos ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

The primary objective of this study was to determine the percentage of individual, preimplantation, in vitro-produced bovine embryos which maintained association with virus despite washing following artificial exposure to a high affinity strain of bovine viral diarrhea virus (BVDV). Another objective of this study was to determine the quantity of virus associated with these embryos. A total of eighty-seven zona pellucida-intact, Day 7, in vitro-produced bovine embryos were exposed for 1 h to 2 � 106 cell culture infected doses per mL to the 50 percent endpoint (CCID50 mL–1) of a type 1 noncytopathic strain of BVDV (SD-1). Following exposure, the embryos were washed according to International Embryo Transfer Society standards for in vitro-produced bovine embryos; they then underwent sonication, RNA extraction, and freezing at –80�C until assayed for virus. A real-time quantitative polymerase chain reaction (QPCR) was run in duplicate on each of the 87 embryos. Forty-two percent (39/87) of the embryos assayed were determined to be positive for virus. The quantity of virus associated with the embryos averaged 0.55 viral copies per 5 µL (SD = 0.89 copies/5 µL, SEM = 0.14 copies/5 µL). Assessment of data using tolerance intervals (P = 0.05) indicates that 90% of contaminated embryos were associated with ≤2.40 viral copies per 5 µL while 99% of contaminated embryos were associated with ≤3.44 viral copies per 5 µL. These findings show that there is a low level of virus associated with in vitro-produced embryos but virus is associated with a significant number of exposed embryos. In conclusion, this study indicates that the potential for transmission of BVDV via embryo transfer of in vitro-produced embryos is small given the amount of virus that was found to associate with individual embryos.

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