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Transgenic Core (CHOP)

Director: Harman, Adele

Summary:

When it comes to using a mouse or rat genome to study human disease, you need the best experimental model available to advance your research and propel discovery. That’s where we come in. The Transgenic Core at Children’s Hospital Research Institute can build you complex mouse or rat models, genetically manipulating the mouse or rat genome to meet your specific research needs. This is accomplished by using cutting-edge and classical genetic engineering approaches. We have successfully created over 40 mouse lines using the CRISPR system, and more recently created our first CRISPR Knockout rat line.

The Transgenic Core is a service sponsored by the CHOP Research Institution to enable investigators to drive cutting-edge basic and bench-to-bedside research. The mission of the Core is to provide a cost-effective fast method for generation and preservation of genetically altered mice for the research community.

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Instruments

  • Bio Rad Gene Pulser Xcell Electroporator ( Electroporator )

    This electroporator uses capacitators to produce controlled exponential or square wave electrical pulses for cell penetration. This piece of equipment is used to electroporate CRISPR RNP products into zygotes to create new founder strains.

  • Bio-Cool IV controlled rate freezers ( Freezer )

    These freezers provide a slow controlled rate of freezing in the range of -40oC to -80oC. The freezing time and temperatures can be adjusted for many applications. The core has two controlled rate freezers. The slow freezing protocol has proven to be very reliable and a consistent method for cryopreserving mouse strains.

  • Cook MINC benchtop incubators ( Incubator )

    These small benchtop incubators provide a microprocessor control system, humidified, gassed and stable heated environment for embryo culture. Due to their size and design they have the ability to rapidly recover homeostasis and temperature. The core has two MINC benchtop incubators and they are a crucial tool for performing temperature sensitive services such as in-vitro fertilization. They are strategically positioned next to the stereo microscopes and enable the user to warm and gas their micro-culture dishes faster than traditional CO2 incubators. They also require considerably less space, but do require a mixed gas combination, rather than just a CO2 supply.

  • DMI 3000B Leica inverted microscopes ( Inverted microscope )

    The manually adjustable inverted microscope is designed for transmitting light to aid visualization of mouse sperm, oocytes, zygotes, blastocysts, and ES cells. The core has two inverted microscopes positioned on top of TMC anti-vibration tables. Each scope is equipped with 4x, 10x and 20x DCI optics enabling high magnification of samples. The microscopes are used to support and visualize embryos during the microinjection of CRISPR, DNA or ES cells.

  • Eppendorf Cell Tram air microinjector ( Microinjector )

    This high precision pneumatic manual microinjector is an air-controlled syringe-like piece of equipment. Once a glass capillary has been inserted into the instrument holder the user can position and secure zygotes, or blastocysts during the microinjection process. The core has two Cell Tram air microinjections, one for each microinjection station.

  • Eppendorf Cell Tram oil apparatuses ( Microinjector )

    This high precision pneumatic manual microinjector is an oil-controlled syringe-like piece of equipment. Once a glass capillary has been inserted into the instrument holder the user can select healthy single embryonic stem cells. The system will hold the cells inside the capillary until ready to inject them into blastocysts. The core has two Cell Tram oil microinjections, one for each microinjection station.

  • Eppendorf FemtoJet 4i microinjector ( Microinjector )

    The FemtoJet with its built-in compressor provide regulated pressure to expel DNA transgenes or CRISPR reagents through injection needles into zygotes during microinjection.

  • Hamilton Thorne XYClone laser ( Laser )

    The laser is attached to one of the inverted microscopes and is used for Assisted-IVF to create a hole in the zona pellucida of each oocyte, thereby facilitating sperm entry and fertilization.

  • Leica left and Leica right manual manipulators ( Micromanipulator )

    These are considered the ‘arms’ of the microinjection process. They control the fine movements that are a critical component for manipulating and microinjecting zygotes or blastocysts.

  • MVE 1400 Series liquid nitrogen storage units ( Liquid nitrogen dewar )

    MVE 1400 Series liquid nitrogen storage units
    These large liquid nitrogen storage units have a large capacity, precise microprocessor control monitoring system, auto-fill system and a wide neck opening for easy access to all samples. The core has two units filled with custom made racks for housing cassettes and straws. Samples in these units are maintained in liquid phase of nitrogen, rather than vapor-phase.

  • MVE XC47/11 liquid nitrogen storage unit ( Liquid nitrogen dewar )

    This small easy maintenance storage unit has the capacity to store long insemination straws and cryovials that are attached to 12” canes. The canes sit inside one of six goblets and are stored in the liquid phase and have the potential remain viable indefinitely. The long neck of the canister reduces liquid nitrogen loss and improves vacuum performance. Samples in these units are maintained in liquid phase of nitrogen, rather than vapor-phase.

  • MZ6 Leica stereo microscope ( Dissecting surgical microscope )

    These stereo microscopes have a 0.63x to 4.0x zoom range, 10x eyepieces are modular and can be adapted to accommodate a camera. The light source is provided underneath the working stage. They are primarily used for micro-dissections and aid visualization of preimplantation stage embryos. They are a key component of the daily core work and have many uses.

  • Surgical MZ6 Leica microscope with KL 1500 LCD light source and swing-arm ( Surgical microscope )

    Leica MZ6 microscopes have a 0.63x to 4.0x zoom range, 10x eyepieces are modular and can be adapted to accommodate a camera. The LCD light source is provided from above the working area and coarse or fine motor focus. The swing-arm configuration helps to expand the range of the stereo microscope and to move across a large work specimen. The core has two microscopes with the same configuration enabling simultaneous use for perform precision micro-surgery.

  • Sutter Instruments P-97 Puller ( Micropipette puller )

    The P-97 is used to pull glass capillaries for multiple applications, such as microinjection needles or holding pipettes. This instrument has many parameters, which can be adjusted, to create custom fine-tip needles for many specific research modalities. The core uses the P-97 puller to create its DNA/RNA microinjection needles and holding pipettes, used to secure a single embryo during the injection procedure.

  • TMC Anti-vibration table ( Vibration isolation table )

    Active vibration isolation tables with rigid steel frame constructed in a desk-style configuration. The core uses them for the demanding microinjection of pre-implantation stage embryos. They are designed to damper movements, that would otherwise not be noticed in typical laboratory settings. They are 3 ft x 4 ft and provide enough working space to accommodate an inverted microscope with manipulators.

  • TPI microforge ( Microforge )

    The microforge offers a range of functions such as cutting, bending, fire-polishing and forming a spike. The thin filament can be heated and cooled as necessary. Rotation mechanisms allow the manual movement of glass capillaries. Positioning the capillaries near the heated filament will determine the shape and size of the end product. The core has a single micro-forge and routinely use it to greatly reduce the opening of pulled glass capillaries. These capillaries are then repositioned to produce a 20 degree bend approximately 5mm from the opening. The small opening prevents zygotes from being taken-up into the pipette and the smooth ends prevent cellular damage.

Services

  • Embryo Cryopreservation ( Material production service )

    Cryopreservation provides a useful tool for archiving mouse lines. The reasons to cryopreserve strains include:

    -- Protect against the loss of valuable strains through breeding failure, disease, human error, etc.
    -- Eliminate the cost of maintaining inactive mouse lines.
    -- Free up space for other mouse lines.
    -- Facilitate future rederivation into a new facility.

    Frozen embryos can be stored indefinitely and thawed many years later, should the investigator require the use of the archived mouse line.

    Archiving a complex, involves more than two mutant alleles, mouse line is accomplished by freezing a minimum of 250 embryos. This method necessitates the use of 10-20 of your young male mice mated, overnight, with 10-20 of your female mice. The following day, viable 2-cell embryos are isolated and cryopreserved. The straws, containing the frozen embryos, are held in the Core large storage unit until needed. Viability of your cryopreserved line is performed by thawing a sample of embryos and culturing to the blastocyst stage. If requested, we can also surgically transfer some of the thawed embryos to generate live mice, but this would involve additional service fees.

  • IVF for Embryo Cryopreservation ( Material production service )

    Investigators who require a strain to be frozen using embryos, are often confounded and inhibited by the number of mice needed to complete this task. To overcome this hurdle, we have the ability to perform a large IVF service. In doing so we are able to generate the desired number of embryos requested to secure the strain. This can be completed using wild-type oocyte donor females, or mutant oocyte donor females, and sperm from one of your males. If using your donor females, we would need ten young (3-5 weeks old) mice. Males supplied for this service should always be individually housed, healthy and a minimum of 12 weeks old.

  • Mouse CRISPR/Cas9 mRNA Microinjection ( Material modification service )

    The conventional and traditional method for creating gene targeted mice was to induce a site-specific mutation in mouse embryonic stem (ES) cells, then microinject the ES cells into blastocysts (see ES cell Microinjection). This work is extremely laborious and expensive. Genetic modification technology has evolved and the bacterial method of genetic repair has been adopted, as a faster and cheaper method for creating gene target mutations in animals. This newly adopted system is referred to as the CRISPR/Cas9 method. This technology consists of two main components; the guide RNA (gRNA) that binds to DNA and the enzyme Cas9, which cuts the DNA at a specific location. Once a cut has been made it is possible to:; insert, delete, or alter a specific sequence of interest.
    Microinjecting Cas9 mRNA into zygotes is the preferred method for large gene sequence modifications or conditionals mutations. While some labs may prefer to prepare their own CRISPR reagents, we can also recommend utilizing the Penn School of Medicine CRISPR/Cas9 Mouse Targeting Core or ordering the guides through a commercial vendor such as IDT.
    While success rates may vary for each gene target region, there have been no lines that we were unable to generate founder animals. The average success rate for microinjections is 30%.
    Your mRNA will be injected directly into the cytoplasm of a minimum of 150 fertilized 0.5d.p.c. zygotes. This can be performed in a wild-type strain, or existing mutant strain. The mRNA is loaded into the ultra-fine injection needle, and using a constant, regulated flow of pressure is injected into each zygote. Following an incubation period, the surviving zygotes are surgically transferred into the reproductive tract of 0.5day pseudo-pregnant surrogate females. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for integration of the mRNA sequence. Mice identified with the correct sequence are considered the founders, or F0, these will be transferred to your animal holding room, where they can be bred to expand the line for experimental use.
    The facility will schedule your strain production requests on a first-come, first-served basis. We will attempt to accommodate any strain background requests, but please be aware that hybrid strains tend to work better than inbred strains.

  • Mouse CRISPR/Cas9 RNP electroporation ( Material modification service )

    The conventional and traditional method for creating gene targeted mice was to induce a site-specific mutation in mouse embryonic stem (ES) cells, then microinject the ES cells into blastocysts (see ES cell Microinjection). This work is extremely laborious and expensive. Genetic modification technology has evolved and the bacterial method of genetic repair has been adopted, as a faster and cheaper method for create gene target mutations in animals. This newly adopted system is referred to as the CRISPR/Cas9 method. This technology consists of two main components; the guide RNA (gRNA) that binds to DNA and the enzyme Cas9, which cuts the DNA at a specific location. Once a cut has been made it is possible to:; insert, delete, or alter a specific sequence of interest.

    Electroporation of the Cas9 RNP is typically employed for all mutations such as small insertions, deletions or sequence changes. While some labs may prefer to prepare their own CRISPR reagents, we can also recommend ordering the guides through a commercial vendor such as IDT.

    The RNP will be electroporated with 0.5day gestation zygotes. The electroporation is performed using a Gene Pulser Electroporator and 1mm cuvettes. This procedure allows the RNP complex to pass through the chemically thinned zona pellucida, and penetrate the fertilized zygote where it incorporates into the mouse genome. Shortly after electroporation the zygotes are surgically transferred into the reproductive tract of 0.5day pseudo-pregnant surrogate females. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for integration of the RNP sequence. Mice identified with the correct sequence are considered the founders, or F0, these will be transferred to your animal holding room, where they can be bred to expand the line for experimental use.

    The facility will schedule your strain production requests on a first-come, first-served basis. We will attempt to accommodate any strain background requests, but please be aware that hybrid strains tend to work better than inbred strains. A minimum of 90 electroporated zygotes are transferred into recipient mother mice, the majority of which will become pregnant.

    A cost saving can be offered if 2 lines are generated during the same electroporation session. Both lines can be for your own lab, or you can split the service fee with another investigator.

    If the core does not generate any founders from the first electroporation session a second session will be scheduled, the fees associated with the additional animals will be billed to you, but the core will absorb the service fee. If no founders are identified from the second set of electroporations we will need to arrange a meeting to evaluate the project.

  • Mouse DNA microinjection ( Material modification service )

    Your DNA will be injected directly into the pronucleus of a minimum of 150 fertilized 0.5d.p.c. zygotes. This can be performed in a wild-type strain, or existing mutant strain. The DNA is loaded into the ultra-fine injection needle, and using a constant, regulated flow of pressure is injected into each zygote. Following an incubation period, the surviving zygotes are surgically transferred into the reproductive tract of 0.5day pseudo-pregnant surrogate females. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for integration of the exogenous DNA sequence. Mice identified with the correct sequence are considered the founders, or F0, these will be transferred to your animal holding room, where they can be bred to expand the line for experimental use.

    The facility will inject your DNA construct on a first-come, first-served basis. We will attempt to accommodate any strain background requests, but please be aware that hybrid strains tend to work better than inbred strains. A minimum of 100 microinjected zygotes are transferred into recipient mother mice, the majority of which will become pregnant.

    We expect to generate 30-50 pups from a microinjection session, this number is typically sufficient for the production of founder mice. Although it is common that founder mice can be generated from fewer animals too.

    If the core does not generate any founders from the first microinjection session a second session will be scheduled, the cost of the additional animals will be billed to you, but the core will absorb the service fee. If, however, no founders are identified from the second set of microinjections we will need to arrange a meeting with the principle investigator to discuss the project.

    PREPARATION OF TRANSGENE DNA FOR MICROINJECTION
    General Considerations
    DNA samples for microinjection should be free of contaminants that might harm the single cell zygotes. Such potential contaminants include traces of phenol, ethanol or enzymes. It is also essential to remove any particles that could clog the injection needles. When purifying the DNA, please use powder-free gloves to avoid particulate matter that may interfere with the microinjection. Be aware that autoclaved tubes can have residue in them- ; use sterile cryovials as an alternative.
    The Transgenic Core Facility recommends that all solutions used to prepare DNA, be filtered through a 0.2micron filter. Sterile, embryo-tested water (Sigma W-1503, 500ml) or Milli-Q should be used in all solutions.

    Once you have isolated your injection construct and diluted it to the correct concentration for injection, a final pass through a 0.2micron syringe filter (Millipore, Cat. No. SLGVL04) will remove any remaining impurities that may clog the injection needle. This step is essential.

  • Mouse Embryonic Stem Cell Microinjection ( Material modification service )

    Knockout mouse models are widely used to study human diseases caused by the loss of gene function. Examples of these diseases include cystic fibrosis, beta-thalassemia and various forms of cancer. Information from studies with knockout mice can lead to a better understanding of the pathogenic mechanisms of human genetic and infectious disease, as well as provide animal models that can be used to test new genetic and drug therapies to treat these disorders.

    This artificially induced mutation is carried in every cell of a knockout mouse throughout development. The resulting phenotype (appearance, biochemical characteristics, behavior, etc.) may provide some indication of a gene's normal role in the mouse.

    Knockout mice are produced by a technique called gene targeting. This technique works by isolating and activating a gene sequence and then replacing it with a version of the same gene sequence that contains a mutation. The replacement occurs by homologous recombination, where two very similar DNA sequences line up next to each other and exchange parts. Gene targeting is carried out in mouse embryonic stem (ES) cells. Embryonic stem cells are derived from very early (usually male) mouse embryos and have the capacity to contribute to the complete development of the animal. The aim is to get modified ES cells to contribute to a germ line. Some sperm are produced that carry the desired mutation and; these will then fertilize a wild-type egg. The resulting progeny develop with one copy of the mutated gene in every cell. Interbreeding these offspring will generate some homozygous individuals that carry both copies of the mutated gene – these are the knockout mice.

    Prior to initiating chimera production, investigators must ensure they have provided documented evidence that the parental cell line has been screened for absence of microorganism contamination. It is important to remember that feeder cells (primary mouse embryonic fibroblasts, MEF) must also be screened for the presence of pathogens; these also have the potential to transmit pathogens to the ES cells. Additionally, the clones, containing the correct target sequence will need to be karyotyped to ensure they contain the correct number of chromosomes, to ensure they are not euploid.

    (A) Normal Session: Chimeric mice will be produced by injecting Embryonic Stem (ES) Cell clones, containing a specific gene targeted mutation, into the blastocyst cavity of E3.5day embryos. The injected blastocysts are cultured 1-3 hours and then surgically transferred into the uterus of a 2.5d.p.c. pseudo-pregnant recipient mother mouse. Approximately 10 days after birth the pups are identified for level of cell contribution, which is apparent by the coat color differences. The fur of chimeric heterozygous pups will be a mix of the host blastocyst strain and the ES cell strain. Mice with the highest amount of cell-derived fur will be selected for future breeding to establish the colony. These mice will be transferred to you animal holding room, where they can be bred to check for germline transmission and to expand the line for experimental use.

    (B) No ES Cells: Occasions may arise when the ES cells being, prepared for microinjection, are not viable, either due to contamination or growth-related problems. In this instance a lower fee has been developed, which will cover the cost of the work performed up-to the day of the microinjections. If the project needs to be repeated you will incur the full-service fee.

    (C) Debris with ES cells: Often when the Transgenic Core receives ES cells, for microinjection, they contain lots of debris, either from dead cells or feeder cells. When this occurs, the debris obstructs access to the viable ES cells, sticks to the outside of the injection needle and will also clog the injection needle. These problems lead to a much lengthier microinjection day and also the use of more, than typical, injection needles. This additional timing and extra supplies are reflected in the increased fee structure for this service. The above problems do not inhibit the project from being completed, they just slow the process down.
    The facility will schedule your strain production requests on a first-come, first-served basis. We will attempt to accommodate any appropriate strain background requests. The core will microinject approximately 40-50 blastocysts for each ES cell line. Approximately 8-15 ES cells are microinjected into each blastocoel cavity. If available the transgenic core will microinject up to 2 clones of the same targeted line per session.

    We expect to generate 10-20 pups from an ES cell microinjection session, this number is typically sufficient for the production of chimeric mice. Although it is common that founder mice can be generated from fewer animals too. Germ line transmission of chimeric animals cannot be guaranteed.

    If the core does not generate any founders from the first microinjection session a second session will be scheduled, the fees associated with the additional animals will be billed to you, but the core will absorb the service fee. If, however, no founders are identified from the second set of microinjections we will need to arrange a meeting with the principle investigator to evaluate the project.

  • Mouse Line Rederivation ( Material production service )

    Mouse line rederivation is used in many institutions as a method of re-establishing you mouse line or as a means to eliminate pathogenic or potentially pathogenic agents. This procedure involves transferring pre-implantation stage zygotes into recipient surrogate mothers (for completion of development). Fertilized zygotes for rederivation can be generated in several different ways, described below:

    –Harvest and freeze embryos; This option is performed for complex (more than 2 mutant alleles) mouse lines housed in ARC, that do not possess the required health status to enter the CTRB animal facility. Your males (minimum of 5 mice, 7 weeks old+) are mated with your females (5 mice, 3-5 weeks old), the resulting fertilized zygotes are harvested and cryopreserved. A day or two later the zygotes are thawed and transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use.

    -Pre-frozen embryos; The fastest and most efficient method for importing a mouse line is to have pre-frozen embryos shipped directly to the Transgenic Core. We will receive the shipment, store appropriately until needed and regenerate the mouse line. Alternatively, the strain may already be archived in our storage units. To recover the strain, vials are removed from liquid nitrogen, the zygotes are slowly thawed and rehydrated then the zygotes are transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use.

    -Cooled sperm; This service is ideal for importing strains from institutions who do not have access to cryopreservation facilities. Reproductive tissues from 1-2 male mice are shipped to the Transgenic Core at 4 degrees. Upon receipt of the samples the Core will perform In-Vitro Fertilization (IVF) with oocytes isolated from wild-type donors. Fertilized zygotes are transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use. This service requires a strict schedule, due to the viability of the sperm, and the essential timing involved when performing IVF.

    -Pre-frozen sperm; The pre-frozen sperm can be imported directly to the Transgenic Core. We will receive the shipment, store appropriately until needed and regenerate the mouse-line. Alternatively, the strain may already be archived in our storage units. To recover the strain, vials are removed from liquid nitrogen, the sperm is slowly thawed, allowed to recover then used for IVF with oocytes, isolated from wild-type donors. The following day viable zygotes are transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use.

    -Freeze and thaw sperm for IVF; This option is performed for less complex mouse lines housed in ARC, that do not possess the required health status to enter the CTRB animal facility. Tissues are removed from 1-3 males and the sperm is isolated and frozen. Approximately a week later a sample of the sperm is thawed and used for IVF with oocytes isolated from wild-type donors. Fertilized zygotes are transferred into surrogate mother mice housed in CTRB. When the rederived offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype will be transferred to your animal holding room for future breeding and experimental use.

    The method of rederivation you choose to adopt should be determined based on the genotype of the animals and your goals with the strain. Strains involving 2 or more allele modifications ideally should not be rederived using in-vitro fertilization options. The zygote transfer methods (A or B) would produce mice with the correct genotype much faster than the sperm options.

    All strains of mice can be rederived, but the efficiency of the procedure depends on the hardiness and the fertility rate of the strain. Inbred strains may occasionally require the use of more mice and could take exceptionally more time.

    After the zygotes have been surgically transferred into the reproductive tracts of the surrogate mother mice they will remain in the care of the transgenic core. The surrogates will deliver the pups 3 weeks post-transfer. Approximately 2 weeks later the Core will isolate tail snips for your lab to genotype. The offspring will be weaned when they are 3 weeks old. At this time the Veterinary Department may decide to perform some health assessments on the animals to verify their health status. This procedure will take approximately one to two weeks to complete. After which, the animals can be transferred to your animal holding room.

  • Rat CRISPR/Cas9 mRNA Microinjection ( Material modification service )

    The conventional and traditional method for creating gene-targeted rats was to induce a site-specific mutation in mouse embryonic stem (ES) cells, then microinject the ES cells into blastocysts. This work is laborious and expensive. Genetic modification technology has evolved and the bacterial method of genetic repair has been adopted as a faster and cheaper method for creating gene target mutations in animals. This newly adopted system is referred to as the CRISPR/Cas9 method. This technology consists of two main components; the guide RNA (gRNA) that binds to DNA and the enzyme Cas9, which cuts the DNA at a specific location. Once DNA has been cut it is possible to insert, delete, or alter a specific sequence of interest.

    Microinjecting Cas9 mRNA into zygotes is the preferred method for large gene sequence modifications or conditionals mutations. While some labs may prefer to prepare their own CRISPR reagents, the Transgenic Core Lab also recommends using the Penn School of Medicine CRISPR/Cas9 Mouse Targeting Core or ordering the guides through a commercial vendor such as IDT.

    Your mRNA will be injected directly into the cytoplasm of a minimum of 100 fertilized 0.5d.p.c. zygotes. This can be performed in a wild-type or existing mutant strain. The mRNA is loaded into the ultra-fine injection needle and using a constant, regulated flow of pressure is then injected into each zygote. Following an incubation period, the surviving zygotes are surgically transferred into the reproductive tract of 0.5day pseudo-pregnant surrogate females. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab or the CRISPR Core and screened for integration of the mRNA sequence. Rats identified with the correct sequence are considered the founders, or F0; these will be transferred to your animal holding room, where they can be bred to expand the line for experimental use.

    The Transgenic Core will schedule strain production requests on a first-come, first-served basis. We will attempt to accommodate any strain background requests, but please be aware that hybrid/outbred strains tend to work better than inbred strains. A minimum of 50 microinjected zygotes are transferred into recipient mother rats, the majority of which will become pregnant.

    If the core does not generate any founders from the first microinjection session a second session will be scheduled. The fees associated with the additional animals will be billed to you, but the core will absorb the service fee. If no founders are identified from the second set of microinjections we will need to arrange a meeting to evaluate the project.

  • Shipping and Receiving Frozen Gametes (Strain) ( Material production service )

    Frozen stocks of mouse embryos and sperm are stored in the Transgenic Core large storage units. Often investigators opt to share their unique mouse lines with researchers at other institutions. This service fee covers the time and supplies needed to remove the appropriate samples from the storage unit, coordinate the shipment and transport the samples to the Shipping Core Facility.

    Importing frozen strains is a valuable resource utilized by investigators who need to use unique strains, that are not already housed in the CHOP facility. The external institution will ship the frozen sperm, or embryos, directly to the Transgenic Core. Upon receipt the Core will unpack and store the gametes for future use. The Core will also arrange return of the shipping container.

  • Sperm Cryopreservation ( Material production service )

    Cryopreservation provides a useful tool for archiving mouse lines. The reasons to cryopreserve strains include:

    -- Protect against the loss of valuable strains through breeding failure, disease, human error, etc.
    -- Eliminate the cost of maintaining inactive mouse lines.
    -- Free up space for other mouse lines.
    -- Facilitate future rederivation into a new facility.

    Frozen sperm can be stored indefinitely and thawed many years later, should the investigator require the use of the archived mouse line. The Core will cryopreserve 15 samples (straws) of sperm for each strain. To achieve this, you must supply 3 healthy male mice, minimum of 12 weeks old, the mice will need to be individually housed at least 4 days prior to the scheduled freeze date. The sperm from the males is pooled together for the freezing. If necessary we can freeze the sperm from each male separately, but this will incur additional fees. There are two options for freezing sperm: without in-vitro fertilization, or with in-vitro fertilization.

    Sperm Cryopreservation (without IVF quality control)
    Approximately 2 days post-sperm freezing, a sample is retrieved from the liquid nitrogen unit, it is thawed, incubated, and used to assess the viability of the strain. The viability of the line is recorded and relayed to your lab. If the viability of the sperm is extremely low, a second session of freezing will be required to safely secure the line. However, it is not often that this is needed.

    Sperm Cryopreservation (with IVF quality control)
    Approximately a week post-freezing a single straw of the sperm is used for IVF, with wild-type oocytes. The following day viable zygotes are transferred into surrogate mother mice to reestablish the mouse line. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab and screened for the appropriate genotype. Mice identified with the correct genotype can be transferred to your animal holding room for future use, or euthanized.


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Last updated: 2019-10-16T11:40:39.132-04:00

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