Transgenic mouse genotyping protocol pdf

The optimization was performed on pcr protocols for genotyping the following transgenic mouse lines. Southern blots are commonly used to determine transgene copy number and the number of integration sites in transgenic founder mice. Noninvasive genotyping of transgenic mice altex alternatives. Methods and protocols is a valuable resource for both novice and expert researchers who are interested in. Genotyping protocol for lacz transgenic mice, including. Thermo scientific phire animal tissue direct pcr kit is designed for dna amplification directly from animal tissues.

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. 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. Rapid noninvasive genotyping of reporter transgenic mammals. Accuracy, speed and access to your genotyping data are paramount to proper management of your studies. Slow and laborious genotyping procedures add to the hassle. Pcr method for genotyping and zygositytesting of rash2. 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. 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. Weight dna for reliable genotyping of transgenic mice. 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. With pronuclear microinjection, a predesigned gene or genes are injected and randomly integrated into the mouse or rat genome. Genotyping transgenic mice by pcr university of michigan transgenic animal model core test mice for the presence of the transgene by pcr.

Guidelines for tissue collection for genotyping mice and rats. The transgenic core guarantees that at least three transgenic founder mice or rats will be produced for each transgene dna construct. Thermo scientific phire animal tissue direct pcr kit. Genotyping of putative transgenic mice individual tail samples are incubated overnight at 55oc in 400 ul of lysis. Guidelines for production of transgenic mice by pronuclear injection. Commonly applied genotyping of transgenic mice involves using tail or ear biopsies which may cause discomfort to the animal. 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. This protocol was developed from the nih guidelines for the. 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.

Genotyping protocols for commonly used sma model mice treatnmd. Transgenic mouse production transgenic core, gensat project april 14, 2010 embryos from gonadotropintreated fvbn female mice taconic are. Crawley, author of the first and second editions of whats wrong with my mouse. How can i tell the difference between a homozygous tgtg mouse and a hemizygous tg0 mouse. 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. Realtime quantitative pcr qpcr is a fast, sensitive, specific, and quantitative method for genotyping transgenic animals. In 2001, laragen successfully applied taqman technique to genotype app and ps1 double transgenic mice. Protocol for genotyping knockout mice stephanie kim and guowei sean zuo 102009 i. However, the dna obtained by the hotshot method is extensively sheared, and its concentration is often too low. Random transgenic mice taconics custom transgenics program employs pronuclear microinjection technology to generate geneticallymodified founder animals.

Alpha1 antitrypsin piz genotyping murine pcr dna extraction. A blastocyst was transferred to a thinwall plastic pcr. 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. Pcr protocol provided by donating investigator reagent constituent volume. Noninvasive transgenic mouse genotyping using stool. The transgenic core provides mouse genotyping and embryo freezing services for the research community of the university of mississippi medical center. Pdf realtime quantitative pcr qpcr is a fast, sensitive, specific, and. It is provided for those investigators who are unfamiliar with mouse genotyping and who need to establish a genotyping method. Here, we show that expression levels of the ubiquitously expressed. For bacteria, multilocus sequence typing chapter 14 and rapid snp detection with pyrosequencing chapter 15 are described. There is an easy way to locate genotyping protocols for a specific strain on our website. 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.

Other simplified methods for extracting dna from mouse tissues are available. Here we present protocols for transgenic mice genotyping achieved directly from mouse. 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. B, aortic root section from a transgenic mouse shows a completely occluded coronary artery.

Through an exclusive distributorship with transnetyx, inc. Pronuclear injection is the most widely used protocol for the generation of transgenic mice. 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. The core facility in our university has injected the dna for our transgenic mouse. Because of the sensitivity of pcr, crosscontamination between mice can occur by carryover on the punch or forceps.

Genotyping of transgenic animals by realtime quantitative. Genotyping protocols for commonly used sma model mice. 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. The core also provides protocols and reagents for the isolation of. 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. 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. Behavioral phenotyping of transgenic and knockout mice,continues to field calls and emails from molecular geneticists who ask. 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.

Genotyping protocol for the alpha1 antitrypsin piz mouse model. Tissue collection for genotyping mice and rats, 022818. Genotyping protocol for the alpha1 antitrypsin piz. Universal mouse genotyping protocol mouse genetics core.

Genotyping protocol for mef2c73kcre and mef2cahfcre. Here, we describe all steps involved from dna purification to the set up of a mouse colony including. Transgenic mouse production transgenic core, gensat. 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 genotyping protocol presented here is very robust in most cases. Pronuclear injection for the production of transgenic mice. Animal research advisory committee guidelines oacu. Generic egfp genotyping protocol for gensat bac transgenic strains protocol. The protocol is flexible and can be optimized for individual.

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. Transgenic mice custom transgene expression taconic. Tcr transgenic mice reveal stepwise, multisite acquisition of the distinctive fattreg phenotype. A transgenic mouse model exists that expresses the mutant human piz aat gene. For those familiar with pcr genotyping, this method differs from the typical protocol by utilizing a unique enzyme.

Basic protocol 1 tissue sampling methods and procedure there are several ways to obtain dna for mouse genotyping. Homozygous and hemizygous transgenic mice can be distinguished using one of three techniques. Laragens unique knowledge and experience with mouse genotyping stands us apart from others. Last, but certainly not least, it is critical for genotyping findings to be reported in a clear and unambiguous fashion. Thus, hotshot dna preparations are likely to be limited to pcr applications and are not suitable for genotyping transgenic mice by southern analysis. Dna in crude extracted from tail, ear, or toe tissue, with speci. Genotyping of the tg mice was performed by polymerase chain reaction. Pdf noninvasive instant genotyping of fluorescently. There are several ways to obtain dna for mouse genotyping. Euthanasia of rodents using carbon dioxide, 102319 revised. 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. Inherent to breeding genetically modified mice and rats there is the need for regular genotyping.

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. Transgenic mice should be genotyped across a unique junction fragment. In short, generating transgenic mice involves five basic steps. Publications supported by the transgenic core this is how we test mice and rats for the presence of the transgene by pcr. The core provides interested investigators help in the design of pcr primers for genotyping of transgenic or genetargeted mice. Rapid and effective genotyping of cre transgenic mice. 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. Guidelines for acceptable methods of identification and.

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. Protocol for genotyping knockout mice university of chicago. However, the choice of primers will differ depending upon the method used to generate the model. For pcr assays, test the specificity and sensitivity.

Optimization of pcr protocols used for genotyping transgenic mice. Find the data sheet of a strain of interest by stock number catalog number, by gene name or by strain name select the. 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. Transgenic core university of mississippi medical center. What are different ways of genotyping a transgenic mouse. 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. Western analysis of apo ai in plasma and aortic extracts, using a. Genotyping by sequencing for plant analysis is also included chapter 16. Genotyping genetically modified gm mice springerlink. A noninvasive ocular tear sampling method for genetic. Transgenic core facility genomic sciences and precision.