Briefly Describe How To Transfer Files From Your Culture Vial To The Knockout Vial

Mouse Embryonic Stem Cell Culture Procedures & Protocols

When transgenic and gene knockout techniques were developed, they provided a powerful tool for the investigation of gene function. The capacity to extract and cultivate mouse embryonic stem (ES) cells in vitro has shown to be critical in this process. ES cells, which are derived from the inner cell mass of early mouse embryos, contribute to the development of all tissues, including germline tissue. The development of efficient protocols for the in vitro cultivation and maintenance of mouse pluripotent stem cells (ES cells) has been critical to the success of gene targeting studies.

These products include the ESGRO mLIF supplement, primary mouse embryonic fibroblasts, mouse embryonic stem cell lines, stem cell screened FBS, and proprietary serum-free media, among other things.

Remember that the protocols given in this handbook are intended to be used as a reference only, and that optimizing culture procedures is strongly recommended in order to achieve success.

Experimental Outline for Targeting ES Cells –Step by Step

Create a flow chart that spans the period from day 1 to days 16–19. It should be noted that, depending on the development of the ES cells, the days may need to be adjusted. In all, the electroporation process will take 2–3 weeks, including weekends, as would the screening and selecting of DNA samples.

Plating PMEF Feeder Cells

EmbryoMax ®Primary Mouse Embryo Fibroblasts (PMEF) feeder cells are given in frozen vials containing 5–6 x 106 cells per vial at passage 3 (2–3 population doublings per passage) at passage 3 (2–3 population doublings per passage). It is advised that PMEF feeder cells be plated one day before ES cells are plated. This ensures that the PMEF cells are around 95 percent confluent when the ES cells are plated. The feeder layer may have some tiny gaps if ES cells are plated more than one day after PMEF plating.

It is not suggested to plate ES cells while gaps are present, despite the fact that this may not have any negative consequences for the ES cells.

  1. Preparation of plates and flasks with Gelatin solution (Section III) prior to freezing PMEF cells. Thaw the PMEF vial(s) immediately in a 37 °C water bath and transfer to a 15 mL tube (which has previously been pre-filled with 10 mL of warm PMEF Feeder Cell Medium) as soon as possible. Using a gentle inversion motion to spread the contents, centrifuge at 300xg for 4–5 minutes. Remove the supernatant and resuspend the cell pellet in warm PMEF Feeder Cell Medium
  2. Repeat the procedure twice more. To aliquot PMEF feeder cell suspension, remove the Gelatin solution from the plates/flasks and distribute it according to the densities specified in Table 4.1 on the next page. Incubate the PMEF Feeder cells at 37 degrees Celsius with 5% CO2 for 30 minutes. To get an idea of the right PMEF density and look, refer to Figures 4A, B, and Cas as a guidance. Plates that have been gelatinized can be used for 12–14 days.

ES Cell Culture using ESGRO ®Medium Supplement

ESGRO ®supplement is a proprietary formulation of the mouse LIF protein (Lipoprotein IFN). The biological activity of each batch of ESGRO®supplement is measured, as opposed to the weight of ordinary LIF, which is sold by weight. There are several advantages to using ESGRO®mLIF media supplement, including constant suppression of mouse embryonic stem cell differentiation, no batch-to-batch variance, and the ability to culture mouse embryonic stem cells in feeder-free settings.

ES Cell Culture without PMEF Feeder Cells

  1. Thaw a vial containing 1×10 7ES cells into 4 mL of ES Cell Medium (with 1000 units of ESGRO ®supplement per mL) and add it to a culture dish. Centrifuge the cells for 3–5 minutes before resuspending them in 10 mL of ES Cell Medium. Plate the ES cells onto the gelatinized plates at a density of 1–1.5 x 10 6cells/25 cm2 (about 3 x 10 6cells/100 mm plate) to achieve a cell density of 1–1.5 x 10 6cells/25 cm2. Incubate the plates at 37 degrees Celsius with 5% CO2 for 30 minutes. The look of the cells should be similar to Image 5A
  2. Examine the cells on a daily basis to see if a change in medium is necessary (indicated by a change of media color to yellow). A huge number of colonies will form in ES cell cultures after 2–3 days, and the culture will become congested (Image 5B). The ES cells should be passaged one to five times
  3. To transit ES cells, prepare two 100 mm gelatinized plates in preparation as mentioned previously (Section III). Remove the ES Cell Medium from the plates and wash them twice with DPBS before adding 1.2 mL of Trypsin. Plates should be incubated at 37 °C for 2 minutes before being filled with 10 mL of ES cell medium. Break up the ES cell aggregates by pipetting vigorously (prevent the creation of bubbles) and transfer 2 mL of the ES cell suspension to each gelatinized plate containing 8 mL of ES Cell Medium (avoid the formation of bubbles). Excess ES cells can be frozen at a concentration of 2–10 x 10 6 cells per vial for later usage at a concentration of 2–10 x 10 6 cells per vial. Note that ES cells should always be passaged the day before you wish to electroporate
  4. Otherwise, they will not work.

Electroporation of ES Cells

  1. Prepare 4 plates containing PMEF cells (Section IV) the evening before the electroporation procedure is to be done. Feed the ES cells with new ES Cell Medium the morning before the electroporation procedure is to be done. Later that afternoon, harvest the ES cells in the manner outlined earlier and count the number of cells harvested. Electroporation requires a minimum of 1×10 7ES cells, which is the bare minimum amount of ES cells. As previously stated, if there is an excess, the cells should be frozen down
  2. Centrifuge the cells that will be used for electroporation at 300xg for 10 minutes, then aspirate the medium out of the cells. Prepare an electroporation buffer solution in which to suspend the ES cell pellet. It is recommended that 25–40 g of knockout construct DNA (purified) be linearized, ethanol precipitated, and dried as a pellet before starting. The DNA pellet should be dissolved in 30 milliliters of Electroporation Buffer before being added to the ES cells. This should be done in a clean environment. Combine together and set aside for 5 minutes at room temperature. Place the ES cells in an electroporation cuvette with a diameter of 0.4 cm. Electroporate the suspension at 500 FD and 0.24 kV for 10 minutes. There should be a time constant created that is between 6.9 and 7.9 milliseconds in length (optimal 7.2). After electroporation, place the cuvette on ice for 10 minutes to cool it down. To mix gently, use a Pasteur pipette to transfer the electroporated ES cells to 40 mL of ES Cell Medium. Placing the ES cell suspension on a plate (10 mL per feeder plate, total of 4 plates). Check to verify that all of the PMEF Feeder Cell Medium has been withdrawn before adding any more cells. Prior to antibiotic selection, incubate the samples for about 36 hours at 37 degrees Celsius and 5 percent CO2.

Selection of ES Cells

In order to establish the correct medication concentration to be employed in each ES cell line, it is advised that a death curve be developed for each ES cell line before selection.

As a general guideline, the following selection regimes can be used:

  • Neomycin (G418) was used in the following concentrations in 129SVEV and 129SVJ cells: 350 g/mL for two days, then 150 g/mL for the remainder of the selection. Selection took 5–7 days in total
  • Neomycin (G418) was used in C57BL6/J cells at concentrations of 275 g/mL for one day, 200 g/mL for the second day, and 150 g/mL for the remainder of the selection. Selection took 6–8 days in total
  • H ygromycin B was used in 129SVEV and 129SVJ cells at concentrations of 100 g/mL for two days, 75 g/mL for one day, and 50 g/mL for the remainder of the selection. Selection takes a total of 7–10 days.
  1. After 48 hours, the presence of cell death should be evident in ES cell media with either Neomycin (G418) or Hygromycin B. If there is excessive debris visible or if the medium is discoloring, change the culture media on a daily basis
  2. Otherwise, changing the medium every other day is acceptable. It is necessary to wash the cells with sterile DPBS before changing the medium if the debris is clinging to the living cells
  3. Nonetheless, it is important not to dislodge or disturb the feeder cell layer. Following electroporation, ES cells should be cultivated for roughly 10 days before being used.

Colony Picking

ES cell colonies are normally available for harvesting 5–10 days after electroporation, depending on the cell line and the medium employed. The most acceptable colonies to pick are those that have a rounded or oval form, have a brilliant edge with phase contrast, and typically have a black necrotic core. Differentiated colonies are flat, and they are frequently surrounded by fibroblast-like cells that create cobblestone-like formations in the surrounding area. When picking cells, it is best to stay away from these areas.

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It is not necessary to employ feeder cells if gelatinized plates are used instead, as detailed in the process below.

  1. The day before selecting ES cell colonies, cover an appropriate number of 24-well plates with PMEF Cells (Section IV) or gelatin (Section III) as described in Section III. Make sure you are properly protected when picking ES cell colonies by donning gloves, a gown, and a face mask. All surfaces, including the microscope, the bench, the tip boxes, and the pipette, should be cleaned with ethanol before each usage. Examine the ES cell cultures using a 4x magnification lens. Colonies that are selected for picking should be placed far enough apart from one another to prevent contamination from adjacent colonies. When a desirable colony is discovered, a pipette tip should be used to circle the colony in order to release the surrounding fibroblast layer. Scrape the colony with the pipette tip while the pipette is adjusted to a volume of 15 mL to dislodge the colony, and then aspirate the colony (the colony is often visible inside the tip). Transfer the colony you selected to an empty well in a 96-well plate and set it aside. Continue choosing and transferring ES cell colonies to fresh wells, each time using a different tip, until a sufficient number of colonies has been picked. Clones are frequently selected in batches of 48 cells in order to avoid tiredness. In each well, place a single drop of Trypsin and incubate for 2 minutes at 37 degrees Celsius. If necessary, replace the PMEF Cell Media in the 24-well plates with 500 mL of the ES cell media during this time period. 6. Disperse each ES cell colony in the 96-well plate by breaking up each colony with a pipette, taking care not to create excessive foaming. Apply new tips for each well of a 24-well plate (including foam) that contains 500 microliters of ES cell media
  2. Transfer the suspension to the plate. When all of the colonies have been transferred, use a clean pipette tip set to 400 mL to mix each well well. Incubate at 37 degrees Celsius with 5 percent CO2. Feed once a day with a medium containing antibiotics such as Neomycin (150 g/mL) or Hygromycin (50 g/mL)
  3. Within a few days, you should see the appearance of new colonies. It is necessary to disperse colonies that are excessively close to one another using a 1 mL pipettes tip to break up the colonies and scatter the cells (Trypsin is not required as colonies break up very easily). Before harvesting, make sure that each well is completely covered with colonies. Every day after that, change the ES cell media until a decent coverage of colonies in each well has been established (usually 7–10 days).

Troubleshooting Guide

One of the most common issues encountered while creating knockout mice is the difficulty to develop chimeras once a targeted embryonic stem cell clone has been generated. The other issue is the inability to generate chimeras once a specific ES cell clone has been established. Table 25.1 lists a number of typical reasons of ES cell differentiation, as well as advice for how to assist prevent differentiation from occurring in the first place. When examining the shape of ES cell colonies, it is possible to determine whether or not the cells have differentiated sufficiently.

In these kits, you’ll find monoclonal antibodies to ES cell markers as well as reagents for Alkaline Phosphatase detection, which will allow you to distinguish between pluripotent and differentiated ES cells.

Table 25.3 lists some of the likely reasons for the inability to generate chimeras.

Gene Interaction with Drosophila

In introductory genetics, students frequently investigate the effect of a single gene on a single trait. This is rational since pupils must first comprehend the simpler circumstances before moving on to the more sophisticated ones; otherwise, they would fail. Students, on the other hand, might readily get to the conclusion that one gene equals one phenotype. Actually, there are just a few, if any, cases in which this is the case in practice. The color of the eyes in Drosophila is a typical example.

  1. This is a classic introduction task for students.
  2. This is correct, however in Drosophila, there is no one gene that determines eye color.
  3. In Drosophila, the production of eye color is regulated by a huge number of genes, maybe as many as 100.
  4. The ommochrome route is responsible for the production of brown pigments, whereas the drosopterin pathway is responsible for the production of bright-red pigments.
  5. In the drosopterin pathway, homozygous brown is epistatic to all of the genes involved in the route, while in the ommochrome pathway, homozygous scarlet is epistatic to all of the genes participating in the system.
  6. drosopterin signaling pathway Brown eyes are caused by a mutation in the homozygous brown gene that prevents red pigments from attaching to eye granules.
  7. drosopterin signaling pathway Scarlet is a homozygous mutation that prevents brown pigments from attaching to eye granules, resulting in bright-red eyes.

pathway of ommochromes pathway of drosopterin The homozygous condition is caused by the presence of both mutations in the same gene. Both routes are blocked, and there is no pigment in the eyes. pathway of ommochromes pathway of drosopterin Student Worksheet for the Lab

Needed Materials*

171925DrosophilaGene Interaction Set is a collection of genes that interact with one another (2 cultures: brown and scarlet) culture vials and plugs that have been emptied (173076) label for a vial pencils made of sorwax Formula 4-24® Instant Drosophila Medium (173200) FlyNap® Kit (Formula 4-24® Instant Drosophila Medium (173200) (173010) alcohol Index cards to be used as sorting trays for drosophila (173094) Drosophila sorting brushes CarolinaTMDrosophilaManual (in English) (452620) stereomicroscopes Materials that are optional a CarolinaTMDrosophilaManual and a CarolinaTMDrosophilaStand (173030)Our173050DrosophilaCulture Kitcontains 36 vials and plugs with labels, a FlyNap® Kit, Formula 4-24®, sorting brushes, sorting cards, and a CarolinaTMDrosophilaManual.

Safety

When students are undertaking any activity in the classroom or lab, make sure they understand and follow safe laboratory procedures and procedures. Exhibit the right protocol for utilizing the equipment and materials required to accomplish the tasks, and underline the necessity of following the process while using the supplies. Personal protection equipment, such as safety glasses, goggles, gloves, and aprons, should be used as necessary. Students should see you practicing correct laboratory safety procedures, and they should be held accountable for following all laboratory safety regulations.

Procedures

When students are undertaking any activity in the classroom or lab, make sure they understand and follow safe laboratory procedures and practices. Exhibit the right protocol for utilizing the equipment and supplies required to accomplish the tasks, and highlight the necessity of following the process. Protect yourself from harm by using personal protective equipment such as safety glasses, safety goggles, gloves, and aprons when needed. Students should see you practicing good laboratory safety procedures and should be held accountable for following all laboratory safety regulations.

Answer Key to Questions Asked on the StudentLabSheet

  1. Give the genotypes and phenotypes of the parent flies of your cross based on the information you’ve gathered.
Parent Fly Genotype Phenotype
Female bw / bw,st +/ st + Brown eyes
Male bw +/ bw +,st / st Scarlet eyes
Parent Fly Genotype Phenotype
Female bw +/ bw +,st / st Scarlet eyes
Male bw / bw,st +/ st + Brown eyes
  • Give the genotype of the F 1flies that you anticipate to see. The following combinations are possible: bw +/bw,st +/st
  • What other information would you want in order to make an educated guess on the phenotype of the F 1flies? Which alleles are in the majority. The removal of the parent flies is required for several reasons. (Some students may add that they would like to know whether the bw and st are related and whether either is sex linked.) To the point where they will not mate with their offspring and will thus not be included in statistics for the F 1 generation Anesthetize the F 1flies and examine them under a stereomicroscope to determine their characteristics. Fill up the blanks with your observations. What does the fact that all of the F 1flies, both male and female, have red (wild-type) eyes tell you about the alleles bw and st, you might wonder. The wild-type alleles of both flies are recessive to one another
  • Why is it not essential to choose virgin female flies from the F 1 to accomplish this cross? The genotype of all of the flies in the F 1 is the same
  • Draw a diagram of the F 1cross in the area provided here. If you believe brown and scarlet are not connected, construct a Punnettsquare and fill it in with the predicted F 2phenotypes and their ratios. bw / bw +,st +/ stbw +/ bw,st +/ stbw +/ bw,st +/ st 2Punnett Square
  • F 2Punnett Square
Male Female bw +,st + bw +,st bw,st + bw,st
bw +,st + bw +/ bw +,st +/ st + bw +/ bw +,st / st + bw / bw +,st +/ st + bw / bw +,st / st +
bw +,st bw +/ bw +,st +/ st bw +/ bw +,st / st bw / bw +,st +/ st bw / bw +,st / st
bw,st + bw +/ bw,st +/ st + bw +/ bw,st / st + bw / bw,st +/ st + bw / bw,st / st +
bw,st bw +/ bw,st +/ st bw +/ bw,st / st bw / bw,st +/ st bw / bw,st / st

PHENOTYPE F 2RatioPhenotype 3Brown eyes and 9Red eyes 3Scarlet pupils 1A pair of white eyes There may be some confusion at this stage because, although it seems to be (and is) a dihybrid cross, only one characteristic—the color of the eyes—is altered. Students’ misunderstandings about how genes function may be brought to light and challenged during class discussion. Data Samples for the Colors Brown and Scarlet Phenotypes and numbers of flies in the F2F2

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Date Counted Phenotype 1 Phenotype 2 Phenotype 3 Phenotype 4
Total 579 206 178 61
Expected Ratio 9 3 3 1
  1. Are your results consistent with the ratios that you predicted? Explain. The answers will differ based on the data that has been gathered. It is genetically determined whether or not humans can taste the chemical PTC. For example, it is common to see this feature exhibited as the result of inheriting either a dominant gene for taste or a pair of recessive alleles for non-tasting of PTC. In light of your previous observations of eye color in Drosophila, would you consider this hypothesis to be credible? If yes, what is the reason behind this? I’d be a little dubious. Taste, like the other senses, is a difficult quality to master. In order to define how the various types of taste buds form, as well as their sensitivity to diverse chemical stimuli, a large number of genes would be required. Is this a monohybrid or a cross between two hybrids? Please include an explanation for your response. It is referred to as a dihybrid cross due to the involvement of two gene loci.

Student Worksheet for the Lab

Pre-made Human RNF2 (RING2 / RING1B) knockout HeLa cell line (ab264845)

Product quality is guaranteed, and skilled customer service is available. More information may be found here.

Overview

  • Using CRISPR/Cas9, an 8-bp deletion in exon 2 and the insertion of the selection cassette in exon 2 were used to accomplish the knockout.

Passage number

According to the results of the Western blot analysis, the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please examine the attached data photos. The HeLa cell line (human wild-type) is recommended as a positive control (ab255448). Important: A wild type cell line is not automatically included with a knockout cell line purchase; if one is necessary, please use the code WILDTYPE-TMTK1 to include a suggested wild type cell line at no additional cost with your order.

  1. Media that is devoid of serum and supplemented with methyl cellulose includes 8.7% DMSO in minimal essential medium (MEM).
  2. nitial requirements for proper handling are as follows: When the vial is received, it should be kept in the liquid nitrogen vapor phase rather than at -80°C.
  3. 1.
  4. 3.
  5. 4.
  6. The maximum suggested dilution is 20 milliliters (mL).
  7. Determine the cell count and seed cells in an appropriate cell culture flask at a density of 2×10 4 cells/cm in an adequate cell culture flask 2.

The seeding density is provided as a guide only, and it should be tweaked to accommodate particular lab time constraints. 4. Incubate the culture at 37 degrees Celsius in an incubator with 5% CO2. Cultures should be checked on a daily basis. Subculture rules of engagement:

  • All seeding densities should be calculated based on cell counts obtained through approved procedures. A suggested seeding density of 2×10 4cells/cm 2 is used as a guideline. If necessary, a partial medium change 24 hours before the start of the subculture may be beneficial to stimulate development. Cells should be passaged after they have reached 80-90 percent confluence
  • Else, they will die.
  • Because of the unique technology used in its development, this product is subject to limited use licenses from The Broad Institute of Harvard University, ERS Genomics, Limited, and Sigma-Aldrich Company LLC. We recommend that you visit our restricted use license and patent sites for more information on the license and patent.

Properties

  • The following are the results of Amelogenin X D5S818: 11, 12
  • D13S317: 12, 13.3
  • And D7S820: 8, 12
  • D16S539: 9, 10. The following are the results of vWA: 16, 18
  • TH01: 7
  • TPOX: 8, 12
  • And CSF1PO: 9, 10

Mycoplasma free

  • 8.7 percent Dimethylsulfoxide, 2 percent Cellulose, and methyl ether are among the constituents.

Research areas

  • E3 ubiquitin-protein ligase that catalyzes the monoubiquitination of histone H2A’s ‘Lys-119’, hence playing a critical role in the formation of the histone code and gene regulation. The ubiquitination of H2A ‘Lys-119’ provides a unique tag for epigenetic transcriptional repression and is involved in the inactivation of the X chromosome in female mammalian cells. It is possible that this gene is involved in the beginning of both imprinted and randomly inactivated X. The Polycomb group (PcG) multiprotein PRC1 complex is an essential component in the development of many genes, including the Hox genes. The complex is necessary to maintain the transcriptionally repressed state of many genes, including the Hox genes. The PcG PRC1 complex acts by remodeling chromatin and modifying histones, resulting in chromatin that is heritably altered in terms of its expressibility and mutability. The activity of the E3 ubiquitin-protein ligase is increased by BMI1/PCGF4. When it comes to histone H2A, PRC1 acts as the primary E3 ubiquitin ligase on the histone H2A, whereas RING1 may rather operate as an activator of RNF2 and RING2 activity.

Pathway

  • When UBE2D3 is present, the protein becomes polyubiquitinated (in vitro). Auto-ubiquitination results in monoubiquitination.

Cellular localization

  • Nucleus. Chromosome. Female trophoblast stem (TS) cells, as well as developing embryonic stem (ES) cells, were shown to be enriched on the inactive X chromosome (Xi). During the development of TS and ES cells, the enrichment on Xi is only temporary. Although the interaction with Xi is mitotically persistent in non-differentiated TS cells, it is not in differentiated TS cells. UniProt provided the information.

Applications

This assurance applies to the use of ab264845 in the following applications that have been tested with it: Application notes offer recommended beginning dilutions; nonetheless, it is the end user’s responsibility to find the ideal dilutions/concentrations.

Application Abreviews Notes
WB Use at an assay dependent concentration. Predicted molecular weight: 37 kDa.Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.
Notes
WBUse at an assay dependent concentration. Predicted molecular weight: 37 kDa.Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

Images

  • Western blot analysis of the RNF2 (RING2 / RING1B) deletion HeLa cell line (human RNF2) (ab264845) All lanes are open: Anti-RING2 / RING1B / RNF2 antibody (ab181140) at a 1/1000 dilution was used in this experiment. Lane 1: HeLa cell lysate from a wild-type strain Lane 2: Rnf2 (RING2 / RING1B) knockout HeLa cell lysateLysates/proteins at 20 g per lane for Rnf2 (RING2 / RING1B) knockout HeLa cells The procedure was carried out in a decreasing environment. Band size predicted to be 37 kDa The band size that was observed was 42 kDa. What is causing the difference between the actual band size and the projected band size? Lanes 1 and 2 have a combined signal (red and green). Green -ab181140 was discovered having a molecular weight of 42 kDa. Loading Control (ab7291) seen at 50 kDa with anti-alpha tubulin antibody (red coloration). In a western blot analysis of wild-type HeLa cells, ab181140 was revealed to react with RING2 / RING1B / RNF2 proteins. shortened forms and cleaved fragments may be represented by the band seen in the knockout cell line ab264845 (knockout cell lysateab257640) lane below 42kDa. This has not been pursued any further at this time. SDS-PAGE was used to separate the lysates of wild-type HeLa cells and RNF2 knockout HeLa cells. A 0.1 percent TBST solution containing 3 percent nonfat dry milk was used to block the membrane for 1 hour at room temperature. Ab181140 and Anti-alpha Tubulin antibody-Loading Control (ab7291) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution, respectively, to detect alpha tubulin protein. Secondary antibodies were used to develop the blots, which were incubated for 1 hour at room temperature with goat anti-Rabbit IgG H L (IRDye ® 800CW) preadsorbed (ab216773) and goat anti-Mouse IgG H L (IRDye ® 680RD) preadsorbed (ab216776) secondary antibodies before imaging. Human RNF2 knockdown HeLa cell line was used for Sanger sequencing (ab264845) Allele-1 has an 8-bp deletion in exon 2
  • Allele-2 has an 8-bp loss in exon 3. Human RNF2 knockdown HeLa cell line was used for Sanger sequencing (ab264845) Allele-2: Insertion of the selection cassette into exon 2 of the coding sequence

Datasheets and documents

Ab264845 has not yet been cited in any publications in which it is expressly mentioned.

Customer reviews and Q As

In this case, the product number is CCL-81-VHG TM. This STAT1 deletion Vero cell line has a greatly improved virus production capability, making it an excellent candidate for viral vaccine production. Product category is a type of product. Animal cells are a kind of cell. Product typeCell modelOrganisational structure Cercopithecus aethiops is an African cercopithecus. Morphology renal disease involving epithelial tissue NormalApplications Cell culture in three dimensions Vaccine research and development format of the product Storage Conditions for Frozen Foods Liquid nitrogen is in its vapor phase.

BSL 1

Using the latest edition of the Biosafety in Microbiological and Biomedical Laboratories (BMBL) from the United States Department of Health and Human Services, ATCC calculates the biosafety level of a substance based on our risk assessment. In accordance with your organization’s policies and procedures, as well as any other applicable requirements enforced by your local or national agencies, it is your job to comprehend the dangers connected with the material you are working with. Whenever working with vials, the ATCC strongly advises that suitable personal protection equipment be worn at all times.

When the liquid nitrogen is thawed, the conversion of the liquid nitrogen back to its gas phase may result in the vial bursting or the cap blowing off with lethal force, resulting in flying debris.

Required Products

These goods are essential for the appropriate usage of this item and have been proven to be efficient in assisting with its functionality by independent testing. It is possible that the quality and efficacy of the item will be compromised if you use other items.

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General

Applications that are specific Production of viral vaccines:

  • Tests for verotoxin, efficacy, malaria biology, media testing, mycoplasma testing, substrate testing, transfection host, detection of virus in ground beef, and more are all being conducted at the University of California, San Diego.

Characteristics

The number of cells in a vial The volume of the liquid is approximately 2.0 x 10 6 mL. rowth propertiesAdherentAgeadultGenderFemaleKaryotypeAdherentAgeadultGenderFemaleKaryotype This is a cell line that has a low number of chromosomes (hypodiploid). Comments The ATCC CRISPR-Cas9 gene editing method was used to create this STAT1 knockout Vero cell line, which was obtained from the original Vero cell line (ATCC ®CCL-81TM). An 199 nucleotide deletion in the STAT1 gene, spanning the third intron and fourth exon, has been shown to be present in this cell line homozygously.

It is known as STAT1 (Signal Transducer and Activator of Transcription 1), and it is a transcription factor that is essential for the interferon-mediated cellular antiviral response.

Cell line validation has been performed on the Vero.STAT1KO cell line (ATCC ®CCL-81-VHGTM) at the genomic, transcript, and protein bio-functional levels. When compared to its mother cell line, it demonstrates a much higher viral titer as well as a significantly higher virus generation potential.

Handling information

Package unpacking and storage recommendations

  1. Examine all containers for signs of leaking or damage. Remove the frozen cells from their dry ice packaging as soon as possible and store them at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until they are ready to be used

Medium in its entirety Eagle’s Minimum Essential Media, developed by the American Type Culture Collection (ATCC), serves as the cell line’s foundation medium (EMEM;ATCC 30-2003). To complete the growth media, combine the following ingredients in equal parts with the basic medium: to a final concentration of 10 percent Fetal Bovine Serum (ATCC 30-2020) was added to the mix Temperature37°CA 95 percent of the atmosphere is composed of air, while 5 percent is composed of CO2. Procedures for dealing with the situation In order to achieve the greatest level of viability, the vial should be thawed and the culture started as soon as possible after receiving it.

The loss of vitality will occur if the product is stored at -70°C.

  1. By gently agitating the vial in a 37°C water bath, the vial will become thawed. Keep the O-ring and cap out of the water to minimize the potential of contamination. Thawing should be completed in a short period of time (about 2 minutes). Immediately remove the vial from the water bath once it has thawed and disinfect it by dipping it in or spraying it with 70 percent ethanol before using it again. Starting from this moment forward, all surgeries must be performed under strictly controlled aseptic circumstances. To prepare the culture, transfer the contents of the vial to a centrifuge tube containing 9.0 mL complete culture media and spin for 5 to 7 minutes at about 125 xg. Replenish the cell pellet with the full media that has been suggested (see the specific batch information for the culture recommended dilution ratio). During the recovery of the cells, it is critical to avoid excessive alkalinity in the medium being used. It is recommended that the culture vessel with the entire growth media be placed in the incubator for at least 15 minutes prior to the addition of the vial contents in order to let the medium to attain its normal pH. (7.0 to 7.6). pH ranges from 7.0 to 7.6. In an appropriate incubator, incubate the culture at 37°C for 24 hours. When utilizing the medium indicated in this product sheet, it is advised that you maintain a 5 percent CO 2 in air environment.

Procedure for subculturing The volumes specified in this technique are for a 75 cm 2flask; for culture vessels of different sizes, adjust the quantity of dissociation media by a factor of proportionately less or more. For subculturing this product, Corning ®T-75 flasks (catalog430641) are recommended (see below).

  1. Remove the culture media and throw it away. Rinse the cell layer with 0.25 percent (w/v) Trypsin- 0.53 mM EDTA solution for a brief period of time to ensure that all traces of serum containing trypsin inhibitor have been removed. In a flask, add 2.0 to 3.0 mL of Trypsin-EDTA solution and monitor cells under an inverted microscope until the cell layer is scattered (about 20 minutes) (usually within 5 to 15 minutes). Note: Do not disturb the cells by striking or shaking the flask while you are waiting for them to detach in order to avoid clumping together. Dispersing cells that are difficult to detach can be facilitated by placing them at 37°C for a short time. 6.0 to 8.0 mL of full growth media should be added, and cells should be gently aspirated by pipetting. Make a series of aliquots of the cell suspension and place them in fresh culture containers. It is possible to generate cultures with between 2 and 4 x 10 4 viable cells/cm 2
  2. Incubate cultures at 37°C

Maintain cultures at a cell density of between 2 X 10 4 and 1.6 X 10 5 cells per cm2 for an interval of at least one week. 2.Subcultivation Ratio: This is the ratio of subcultivation to the total amount of subcultivation. It is advised to use a subcultivation ratio of 1:3 to 1:6. Renewal frequency: 2 to 3 times per week on average Chemicals used in cryopreservation Complete growth media supplemented with DMSO at a concentration of 5 percent (v/v) (ATCC 4-X)

Quality control specifications

Testing for bacterial and fungal contamination There was no detection. contamination with mycoplasma was not observed Tests of functionality Testing for the presence of the STAT1 gene mutation. The virus was shown to have a higher titer and greater viral generation capabilities when compared to the original Vero cell line (ATCC® CCL-81TM). Time for the population to double It will take around 28 hours.

History

The depositors are ATCC and the year of origin is 2019.

Legal disclaimers

Use that is intended This product is designed solely for use in laboratory research applications. It is not meant for any therapeutic application in animals or humans, nor is it designed for human or animal ingestion, nor is it intended for any diagnostic purpose. Guaranteed for 30 days from the date of shipment, ATCC ®products are warranted for their viability if they are stored and handled in accordance with the instructions on the product information sheet, the website, and the Certificate of Analysis.

  • When it comes to live cultures, the American Type Culture Collection (ATCC) provides the media composition and reagents that have been shown to be effective for the product.
  • If an other medium formulation or reagent is employed, the ATCC’s viability warranty is no longer valid, and the experiment is considered invalid.
  • Exclusions and limitationsThis product is designed solely for use in laboratory research.
  • Any intended commercial usage is forbidden without first obtaining a license from the American Tobacco Control Commission.
  • Citations from scientific publications and patents are supplied only for the purpose of providing information.
  • In order for this product to be delivered, the customer must agree that he or she is solely responsible for and assumes all risk and responsibility associated with the receipt, handling, storage, disposal, and use of ATCC products.
  • By accepting delivery of the product and any derivatives or changes, the customer undertakes that any activity done with the ATCC product and any offspring or modifications will be conducted in accordance with all relevant laws, regulations, and standards.
  • Despite the fact that every reasonable effort is taken to assure the authenticity and trustworthiness of materials on deposit, ATCC is not liable for any damages resulting from the misidentification or misrepresentation of materials on deposit by others.

Please refer to the material transfer agreement (MTA) for further information on the product’s use and applications. The Metropolitan Transportation Authority (MTA) may be reached at

PermitsRestrictions

If the United States Fish and Wildlife Service issues ATCC a valid export permit, the organization’s international shipment of any material derived from species listed by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) is conditional on the organization receiving a valid export permit from the United States Fish and Wildlife Service. This export permission is obtained by ATCC; you are not need to do any action in order to obtain this export licence.

With order to comply with export regulations, the export permission will be included in the shipment.

Before you purchase anything, check with your country’s CITES management authority or the authorities in charge of administering the CITES permit system to see if the item you’re interested in requires a permission to be imported.

References

Horvath CM, Darnell JE Jr., Horvath CM Transcriptionally Active Stat1 Protein is Required for the Antiviral State Induced by Alpha Interferon and Gamma Interferon. J Virol, vol. 70, no. 6, pp. 647-650, 1996. PubMed:8523587 Meraz, M.A., and colleagues It has been discovered that a targeted deletion of the Stat1 gene in mice shows physiologic selectivity in the JAK-STAT signaling pathway. In 1996, Cell 84:431-442 was published. PubMed:860859 BPEC stands for the British Pharmacopoeia Commission (British Pharmacopoeia Commission).

The British Pharmacopoeia Commission published British Pharmacopoeia Appendix XVI B in 2003.

Didier ES and colleagues Identification and characterization of Encephalitozoon (Septata) intestinailis isolates cultured from nasal mucosa and bronchoalveolar lavage fluids of two HIV-infected individuals J.

Microbiol., vol.

3, pp.

PubMed:8563708 The American Public Health Association is a non-profit organization dedicated to public health.

3rd edition The American Public Health Association published a report in 1992 in Washington, DC.

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