Genotyping protocol for mef2c73kcre and mef2cahfcre transgenic mice black lab by southern blot preferred using a 387 bp ncoibamhi fragment from the cre cdna as a probe on an ecorv digest sequence of the probe. Pdf noninvasive instant genotyping of fluorescently. Through an exclusive distributorship with transnetyx, inc. Mice are extremely powerful mammalian genetic model organisms for basic and medical research, but managing a colony of transgenic mice is time consuming and expensive, many times requiring the help of dedicated technicians. These sequences are very frequent in many genetically modified mouse strains, meaning that you will not be able check reliably for animal misidentification critical parameters or genotype double. It is provided for those investigators who are unfamiliar with dna genotyping and who need to establish a genotyping method. The university of michigan transgenic animal core and other cores provide calculations based on one copy per haploid genome, and thus arrive at values twice as high controls for pcr genotyping in the propagation of transgenic mice. Last, but certainly not least, it is critical for genotyping findings to be reported in a clear and unambiguous fashion. Targeted es cells used to generate knockout or knockin mice should be genotyped using primers from a unique marker in the construct and a region outside of the construct. Genotyping protocol for the alpha1 antitrypsin piz mouse model.
Noninvasive genotyping of transgenic mice altex alternatives. Animal research advisory committee guidelines oacu. Other simplified methods for extracting dna from mouse tissues are available. Although the jackson laboratory does not provide genotyping as a service, there are vendors that perform this service that can be found in online searches. Transgenic core university of mississippi medical center. For those familiar with pcr genotyping, this method differs from the typical protocol by utilizing a unique enzyme.
Random transgenic mice taconics custom transgenics program employs pronuclear microinjection technology to generate geneticallymodified founder animals. Pcr method for genotyping and zygositytesting of rash2 transgenic mice hiroshi suemizu, chika kitomaruyama, yusuke sotomaru, tomoyuki ogura, kyoji hioki, yasuyuki ohnishi, and norikazu tamaoki. Genotyping protocol for lacz transgenic mice, including mef2cl8 lacz, mef2cf6lacz and many others black lab by southern blot preferred using a 352 bp fragment, which was amplified by pcr using the primers laczprobe5 5gtggtggttatgccgatc3 and laczprobe3 5. With pronuclear microinjection, a predesigned gene or genes are injected and randomly integrated into the mouse or rat genome. Laragen is the first commercial company to apply realtime pcr technique in transgenic genotyping. It was also common for investigators to euthanize transgenic mice that did not have the needed genotype for a particular research project. The present invention provides a transgenic mouse and an animal model that is used to assay for the inhibition or activation of the cnr2 gene and methods for screening drugs to treat or prevent psychosis, anxiety, depression, autism disorders, drug addiction, parkinsons disease andor alzheimers disease, multiple sclerosis, inflammation, stroke, osteoporosis, scleroderma or. Pcr protocol provided by donating investigator reagent constituent volume. The transgenic core guarantees that at least three transgenic founder mice or rats will be produced for each transgene dna construct. However, the choice of primers will differ depending upon the method used to generate the model.
Thermo scientific phire animal tissue direct pcr kit. It is provided for those investigators who are unfamiliar with mouse genotyping and who need to establish a genotyping method. Basic protocol 1 tissue sampling methods and procedure there are several ways to obtain dna for mouse genotyping. Pdf realtime quantitative pcr qpcr is a fast, sensitive, specific, and. The core facility in our university has injected the dna for our transgenic mouse. Genotyping transgenic mice by pcr university of michigan transgenic animal model core test mice for the presence of the transgene by pcr. Documentation of a genotyping protocol for identifying transgenic founder mice by southern blot analysis or by pcranalysis of genomic dna must be provided by the investigator before initiation of injection experiments. Methods and protocols is a valuable resource for both novice and expert researchers who are interested in. Transgenic mouse production transgenic core, gensat project april 14, 2010 embryos from gonadotropintreated fvbn female mice taconic are. This protocol is designed to detect sequences in the murine genome by polymerase chain reaction amplification, and is adapted from stratman and simon transgenic res. Genotyping genetically modified gm mice springerlink. Guidelines for production of transgenic mice by pronuclear injection. We tested the possibility of polymerase chain reaction pcrbased mouse genotyping using stool specimens from three transgenic mouse lines that overexpress 1018 transgene copies of human keratin polypeptide 18, as compared to genotyping.
Crawley, author of the first and second editions of whats wrong with my mouse. This protocol was developed from the nih guidelines for the. Genotyping protocol for mef2c73kcre and mef2cahfcre. In 2001, laragen successfully applied taqman technique to genotype app and ps1 double transgenic mice. A transgenic mouse model exists that expresses the mutant human piz aat gene. Since 1989 the core has produced more than 18,000 transgenic mouse founders from more than 1,800 transgenes and more than 800 transgenic rat founders from 63 transgenes. The optimization was performed on pcr protocols for genotyping the following transgenic mouse lines. Genotyping of putative transgenic mice individual tail samples are incubated overnight at 55oc in 400 ul of lysis.
For bacteria, multilocus sequence typing chapter 14 and rapid snp detection with pyrosequencing chapter 15 are described. Protocol for genotyping knockout mice stephanie kim and guowei sean zuo 102009 i. Guidelines for genotyping mice and rats background identification of individual genotype for animals in a litter is critical for research and in reducing the number of animals used in a protocol. Behavioral phenotyping of transgenic and knockout mice,continues to field calls and emails from molecular geneticists who ask. How can i tell the difference between a homozygous tgtg mouse and a hemizygous tg0 mouse. The core provides interested investigators help in the design of pcr primers for genotyping of transgenic or genetargeted mice. The protocol is flexible and can be optimized for individual. The core also provides protocols and reagents for the isolation of. Southern blots are commonly used to determine transgene copy number and the number of integration sites in transgenic founder mice. Accurate quantitation of the number of transgenes helps to identify founders and to create and maintain pure lines of transgenic mice, thus reducing experimental variability. Find the data sheet of a strain of interest by stock number catalog number, by gene name or by strain name select the. Genotyping protocol for lacz transgenic mice, including. Generic egfp genotyping protocol for gensat bac transgenic strains protocol. Weight dna for reliable genotyping of transgenic mice.
Rapid noninvasive genotyping of reporter transgenic mammals. Slow and laborious genotyping procedures add to the hassle. A noninvasive ocular tear sampling method for genetic. There is an easy way to locate genotyping protocols for a specific strain on our website. Accuracy, speed and access to your genotyping data are paramount to proper management of your studies. Thus, hotshot dna preparations are likely to be limited to pcr applications and are not suitable for genotyping transgenic mice by southern analysis. Guidelines for tissue collection for genotyping mice and rats. B, aortic root section from a transgenic mouse shows a completely occluded coronary artery. A blastocyst was transferred to a thinwall plastic pcr. Thermo scientific phire animal tissue direct pcr kit is designed for dna amplification directly from animal tissues. Protocol for genotyping knockout mice university of chicago. The accustart ii mouse genotyping kit is designed for fast and easy preparation of pcrready dna extracts and endpoint pcr analysis from tissues such as tail snips and ear punches commonly used for genotyping knockout and transgenic animals. However, the dna obtained by the hotshot method is extensively sheared, and its concentration is often too low.
Request pdf genotyping protocol for the alpha1 antitrypsin piz mouse model the most common alpha1 antitrypsin aat mutant variant is a missense mutation e342k, commonly referred to as piz. Rapid and effective genotyping of cre transgenic mice 082016 3 conclusions the fragments amplified from the cre transgene and from the myogenin control 320 bp and 250 bp, respectively were easily distinguished using the qiaxcel system, allowing. Optimization of pcr protocols used for genotyping transgenic mice. Pronuclear injection for the production of transgenic mice. Tissue collection for genotyping mice and rats, whether performed by dact personnel or investigators, will follow the protocol described here unless alternate methods are described in an iacuc. What are different ways of genotyping a transgenic mouse. Genotyping protocols for commonly used sma model mice treatnmd. Universal mouse genotyping protocol mouse genetics core. Here we present protocols for transgenic mice genotyping achieved directly from mouse. Pcr method for genotyping and zygositytesting of rash2. Transgenic mice should be genotyped across a unique junction fragment.
Genotyping transgenic mice pia kuusisto, pak yang and chas andre, thermo fisher scientific, vantaa, finland abstract thermo scientific phire animal tissue direct pcr kit is designed for dna amplification directly from animal tissues. Genotyping by sequencing for plant analysis is also included chapter 16. Realtime quantitative pcr qpcr is a fast, sensitive, specific, and quantitative method for genotyping transgenic animals. Genotyping of the tg mice was performed by polymerase chain reaction. Publications supported by the transgenic core this is how we test mice and rats for the presence of the transgene by pcr. Inherent to breeding genetically modified mice and rats there is the need for regular genotyping.
Euthanasia of rodents using carbon dioxide, 102319 revised. Guidelines for acceptable methods of identification and. The transgenic core provides mouse genotyping and embryo freezing services for the research community of the university of mississippi medical center. Fluorescence proteins have been useful as genetic reporters for a wide range of applications in biomedical research and are frequently used for the analysis of transgene activity. Pdf genotyping of transgenic animals by realtime quantitative. Building on previous knowledge and expertise, we focused on key analytical challenges highyields of pure genomic dna, taking into account that an ideal method for genotyping of transgenic mice. Alpha1 antitrypsin piz genotyping murine pcr dna extraction. En rapid and effective genotyping of cre transgenic mice.
In short, generating transgenic mice involves five basic steps. We report here that noninvasive ocular tear sampling for genetic ascertainment of transgenic mice can serve as an innovative ethical safeguard for animal welfare, and as a veritable alternative to the surgical tail biopsies, ear puncture, or blood sampling from the weanling transgenic mice. Investigative personnel are responsible for implementing the approved identification and genotyping methods with oversight and assistance from ulam personnel, as needed the principal investigator is responsible for ensuring that all methods of animal identification and genetic sampling that requires removal of tissue are explicitly listed and approved by the. Because genetic monitoring would be a decisive issue in determining the suitability of the mouse pups, the protocol would probably fall into usda pain category c or d. Here, we show that expression levels of the ubiquitously expressed. Noninvasive transgenic mouse genotyping using stool. Genotyping of transgenic animals by realtime quantitative. There are several ways to obtain dna for mouse genotyping. Pronuclear injection is the most widely used protocol for the generation of transgenic mice.
Genotyping protocol for the alpha1 antitrypsin piz. Laragens unique knowledge and experience with mouse genotyping stands us apart from others. Dna in crude extracted from tail, ear, or toe tissue, with speci. Western analysis of apo ai in plasma and aortic extracts, using a. Pcr protocol provided by donating investigator genotyping by pcr protocol mutant mouse regional resource center. Because of the sensitivity of pcr, crosscontamination between mice can occur by carryover on the punch or forceps.
The genotyping protocol presented here is very robust in most cases. Tcr transgenic mice reveal stepwise, multisite acquisition of the distinctive fattreg phenotype. Rapid and effective genotyping of cre transgenic mice. Transgenic core facility genomic sciences and precision. Transgenic mice custom transgene expression taconic. Transgenic mouse production transgenic core, gensat. For pcr assays, test the specificity and sensitivity. Here, we describe all steps involved from dna purification to the set up of a mouse colony including. Genotyping protocols for commonly used sma model mice. Guidelines for production of transgenic mice by pronuclear. Tissue collection for genotyping mice and rats, 022818. In order to clarify the practicality of noninvasive genotyping. Homozygous and hemizygous transgenic mice can be distinguished using one of three techniques.