More recent technologic advances now allow for the direct introduction of mutations into the mouse genome via directed endonucleases (CRISPR/Cas9). This technology has further increased the power and flexibility of modified mouse model generation. These direct genome editing methodologies eliminate the need for ES cell manipulations and chimera production. Furthermore, it has the capacity to generate mouse models in a time frame that significantly outpaces prior approaches with a consequent major acceleration in the timing of critical studies and a commensurate decrease in cost. However, while generating gene knock-out mice are very successful, at present knock-in efficiencies are limited.
CRISPR genome editing system is a RNA-guided endonucleases system, it allows users to design guideRNA which target DNA sequences of interest. In the presence of Cas9 endonuclease, the gRNA directs CAS9 to unwind and cleave the double stranded DNA. The TMII Core can offer to generate KO, and KI mouse lines using CRISPR -Cas9 technology.
Please contact us with your research plan to set up a project strategy with the most updated technology.
Design of CRISPR targets, synthesis and purification of RNA and DNA components is performed by the customer or a company, though prepared according to specifications supplied by TMII Core. Upon receipt of the RNA/DNA construct, proven by documentation to be of high quality and suitable for microinjection the TMII Core will start the procedure. The transgenic mouse facility will superovulate donor mice, harvest single cell embryos and inject RNA/DNA provided by the investigators. Surviving embryos will be implanted into surrogate pseudopregnant CD-1 recipient females and the TMII will care for the mice though pregnancy, birthing and weaning. The mice will be transferred to the investigator, and the investigator will have full responsibility for further breeding, genetic analysis, observation, etc.
Not all constructs microinjected into mouse embryos will produce genetically modified founder animals, as the biologic effects of the mutation may prove deleterious. Furthermore, not all nuclease RNA or DNA will yield the desired mutations. Furthermore, the combination of the locus to be modified plus the type of modification required greatly influences the likelihood of success. For this reason we do not currently offer a service guarantee to produce animals with the desired genetic modification. For investigators who send us materials for microinjection, the only service guarantee provided is that we will inject or electroporate a minimum of 120 fertilized oocytes and transfer surviving oocytes to pseudo-pregnant recipient females.
Direct microinjection of linear DNA into the male pronucleus of a mouse one-cell embryo has been until recently the method most extensively used in the production of transgenic mice. If the foreign DNA integrates into the mouse chromosomal DNA at the one-cell stage, the animal will contain the injected DNA in every cell, including those of the germ line. If, however, integration occurs later, the resulting animals will be mosaic for the presence of the injected DNA. The number of copies of integrated DNA can vary from one to tens of copies, arranged primarily in tandem head to tail arrays.
Transgenic constructs, as restriction digest containing (50 micrograms) of the plasmid, cut so as to release the transgene from the plasmid backbone are to be supplied by the investigator. The cleanliness of the construct is of utmost importance. It should be absolutely free of any phenol, chloroform, ethidium bromide, high salt and ethanol. Even trace amounts will quickly lyse the injected embryos or interfere with the development of the implanted embryos. “Sticky” DNA preps will clog the injection needles and kill embryos. Great care should be taken when preparing the injection mix as the success of the procedure is highly dependent upon the quality of the DNA being injected into the embryos. Documentation of quality of the plasmid, suitable for microinjection needs to be provided by the investigator, such as a photo of an aliquot of the digest analyzed by agarose gel electrophoresis, with the transgene band clearly identified as a requirement.
Upon receipt of the DNA construct, proven by documentation to be of high quality and suitable for microinjection, we will proceed to inject a minimum of 150 fertilized eggs. After overnight incubation, normal 2-cell embryos will be transferred into surrogate pseudopregnant CD-1 recipient females and their pregnancy will be monitored (the gestation period of the mouse is 19-212 days). Two weeks after birth of potential transgenic pups, the project scientist will be notified of the number of pups. Frozen tail biopsies of all weaned pups will be provided to the investigator to identify founder mice incorporating the transgene. The positive founders will be transferred to the care of the investigator. If these studies (with adequate controls) fail to demonstrate the existence of at least one transgenic animal, we will re-inject at least 80 eggs at the cost of additional animals used to the investigator. If, however, no transgenic animal is detected after the second trial, a meeting will be held between the scientist, the Facility Director, and the Technical Director to discuss the DNA construct, the tail biopsy analysis, and further plans to be agreed upon. On average, 230 injected eggs should yield at least one (and usually more) transgenic mice.
Please be advised that many factors can affect the production efficiency of transgenic mice. Such factors include the number of eggs surviving the injection and developing to 2-cell stage; this will determine the number of transfers into surrogate pseudopregnant CD-1 recipient females. It is important to have a DNA preparation pure of any contaminant and at the appropriate concentration so that toxic effects to the eggs or one-cell embryos can be avoided. Furthermore, although every transfer promises a certain number of pups, that number may vary greatly due to embryo death in utero. This lethality may in certain settings be related to the type of DNA construct used.
Our Pronuclear Injection Service guarantees the production of 3 transgenic founders or 50 total pups, whichever comes first. For this service, we first perform two days of DNA injection. After tissue biopsies from the resulting offspring are assayed for the presence of the transgene, additional days of microinjection are scheduled as needed.
Transgenic constructs designed in agreement with the TMII Core will be supplied by the investigator, though prepared according to specifications supplied by TMII Core.
The standard background strain used for the embryos is B6D2 (C57BL/6 x DBA/2J). Embryos are produced by mating B6D2 females to C57BL/6 males, generating potential founder animals which are 75% C57BL/6 and 25% DBA/2J. Other strains can be used as egg donors, but these may necessitate additional fees. Some strains do not respond to superovulating hormones and/or have poor quality embryos and should be avoided if possible. Pure C57BL/6 embryos can be used for injection, but due to poor survival, there will be an additional fee charged.
Upon receipt of the DNA construct, proven by documentation to be of high quality and suitable for microinjection, microinjection will proceed as described above for PNI.
Depending upon the quality of the nucleic acid purification, integrity and construct size, the Core has an approximate 50% success rate of te-PNI transgenesis in all tested strains of mice, and most projects generate between 5 – 7 founder animals with the one-cell embryos injected. The number of copies of integrated DNA usually varies between 1 to 3.
Please contact us with your research plan to set up a project strategy with the most updated technology.
Intracytoplasmic sperm injection based transgenesis (ICSI-Tr), relies on spermatozoa as DNA vectors. Here, spermatozoa are membrane-damaged or demembranated by freeze thawing or by treatment with TritonX-100 and then incubated with linear transgene DNA. The exposed perinuclear theca of the sperm head interacts with the transgene and serves as a carrier. This sperm-DNA complex is then injected into the cytoplasm of MII arrested oocytes and the transgene is incorporated into the embryonic genome, in a passive fashion via the endogenous DNA repair mechanism. As previously observed for pronuclear microinjection, many of the transgenes integrate as multiple concatamerized vector copies.
The TMII core will separate the sperm head from its tail and prepare it for injection. Specifically, the sperm is immersed in liquid nitrogen for 10 seconds, the tails are cut of and the sperm heads are incubated with linearized transgenes. The activated oocyte are then injected with the sperm head, incubated for 5-7 hours after which the presence of or absence of pronuclei in the oocytes will be determined. Once the presence of the pronuclei is confirmed, the oocytes will be cultured until they reached the 2-cell stage (24–30 h after microinjection). ICSI oocytes will then be transferred into the oviducts of 8- to 16-week-old surrogate pseudopregnant CD-1 females at 0.5 days post coitus (dpc). Pregnant females are allowed to deliver and raise their pups. The pups will then be transferred to the investigator.
The success of an ICSI precudure with thawed or fresh sperm can exhibit large variations from strain to strain. Quality assessment of sperm is based either on motility of the provided sperm (at no additional cost) or by in vitro fertilization of oocytes and progression to 2-cell stage embryos. It should be noted that motility does not guarantee viability.