|
Karolinska Center for Transgene Technologies (KCTT), Karolinska Institutet, Department of Cell and Molecular Biology (CMB), Department of Biosciences and Nutrition (»Add to My Favourites)
|
|
The aim of Karolinska Center for Transgene Technologies (KCTT) is to produce transgenic mice by pronuclear injection and ES cell techniques. KCTT is one of the largest transgenic core facilities in Scandinavia.
|
|
|
|
Category: Core facility
Key words: injection, transgene, DNA, recombination, ES, pronucleus, electroporation, chimeric, embryo freezing, industryinterest
Facility website: http://www.kctt.ki.se/
Interest in collaboration with industry: Yes
Description:
KCTT is a non-commercial, non-profit academic core facility and is located at the Karolinska Campus-North (CMB) and South (Novum). We produce transgenic mice by pronuclear injection and ES cell techniques for research groups at the Karolinska Institute and research groups at other academic institutions The transgenic technology is today an integrated part of biomedical research, and the need to generate transgenic animals with specific genetic alterations is steadily increasing. We are therefore happy to inform you that the capacity to produce transgenic mice has been significantly improved at KI, and we would like to take this opportunity to welcome you as potential customers in this core facility.
KCTT is a non-commercial, non-profit academic core facility located at both Novum and KI Campus. This way, we can serve researchers at both sites, and have a security backup in case of infections in one of the production units. KCTT is one of the largest transgenic core facilities in Scandinavia with a yearly number of 60 - 70 constructs for pronucleus injection, around 30 - 40 electroporations of knockout constructs and 40 - 50 injected ko constructs of targeted ES cells.
We provide the following services:
1.
| Transgenic mice generated by the pronuclear injection technique, generated in various genetic backgrounds, depending on the preference of the research group.
| 2.
| Transgenic mice generated by the ES cell technique including electroporation and injection.
|
Infrastructure/methods:
KCTT can offer the following additional services (C: Campus, N:Novum):
 | Pronucleus Injection (C)
|
| Blastocyst Injection (C)
|
| ES cell targeting (N)
|
| Re-Derivation of infected mouse strains (C)
|
| Embryo aggregation (C)
|
| Embryo Freezing (C)
|
| Karyotyping of ES cells (N)
|
Flow chart for the pronuclear injection technique
The pronuclear injection technique is suitable for adding genes to the genome in situations where the endogenous genetic makeup is not important, for example when dominant effects or promoter/enhancer activities are studied. For overexpression, a promoter/enhancer that directs expression to a particular organ or timepoint during development is coupled to the gene or cDNA of interest, and for gene regulation studies, the promoter/enhancer of interest is coupled to a reporter gene.
A purified DNA construct is injected into fertilized mouse eggs at the one-cell stage. DNA integration in the genome is random, and the number of integrated copies ranges from one to more than a hundred. In most cases, multiple copies of the introduced DNA are integrated in tandem at a single chromosomal site. After injection, eggs are transferred into oviducts of pseudopregnant females, where they develop further, and offspring is born approximately 20 days later. Mice that have the transgene integrated in their genome are identified by DNA analysis. For a more detailed account of the pronuclear injection technique, please see Hogan et al., 1994.
More information is provided on our website.
Flow chart for the ES cell technique
The key features of the ES (embryonic stem) cell technique can briefly be summarized as follows. Specific mutations, for example inactivation or modification of a gene, can be introduced into the genome by homologous recombination, which leads to the replacement of the endogenous gene fragment by the introduced, engineered fragment. Because homologous recombination is a rare event, special selection and screening strategies are needed. Usually a specific selectable marker gene is included in the targeting DNA construct.
A targeting construct, which is made from mouse 129 strain DNA, is introduced into totipotent mouse embryonic stem (ES) cells (isolated from 129 mice). Cells that have integrated the introduced DNA in their genome are selected using a drug that kills cells which have not taken up the marker gene. Selected clones are then analyzed by restriction enzyme digestion and Southern blot or by PCR analysis to identify the clones where homologous recombination has occurred. Positive clones are then expanded. ES cells from correctly targeted clones are introduced into mouse embryos at the blastocyst stage by injection, and injected blastocysts are transferred into the uteri of pseudopregnant females. Chimeric (mosaic) mice consisting of cells originating from the blastocyst and from ES cells are born approximately 17 days after transfer. Chimeric mice are then tested for their ability to transmit the introduced mutation to the next generation (germline transmission).
Practical information:
Waiting List
The orders from Karolinska Institute, Karolinska Hospital, Huddinge Hospital and other KI-connected research groups will be treated on a strict "first come, first served" basis, i.e. the place in the waiting list will be based on the day KCTT receives the DNA construct. The aim is that the time between receipt of the DNA construct and injection into fertilized mouse eggs should not exceed 1 month (for the pronuclear injection technique). For electroporations the time between receipt of the DNA construct and the electroporation into ES cells should not exceed 2 months. For the injection of ES cells, targeted by the research groups, the waiting time should not be longer than 6 - 8 weeks.
2.
| Academic groups (non-KI) and industry
|
Like for KI groups, the waiting time for pronucleus injection, electroporation and injection of own targeted ES cells should be in the range of the time given for KI groups. Besides this KCTT can't promise to keep the place in the waiting list in case urgent constructs from KI groups have to be injected. Before changing a date, KCTT will set all efforts to keep the promised schedule.
Ordering service from KCTT
To place an order for the generation of transgenic mice by the pronuclear injection or ES cell technique, the respective ordering forms found on our website should be used and sent to KCTT. The appendices, belonging to the different forms, should be sent along with the application forms. Incomplete applications cannot be accepted.
It is desirable that the research group notifies KCTT as early as possible, to indicate when they would like the project to be carried out. This greatly facilitates the long-term planning for KCTT.
It is normally not possible to pre-book an injection date, only in case of time sensitive experiments have to be done (e.g. taking out embryos at a certain stage) dates can be booked. If a time point for the injection has been set, the DNA construct has to be delivered latest 2 weeks before the date. If the DNA is not delivered in time or the injection is cancelled too late, the costs for the whole injection date will be charged to the group.
Prices
KCTT is working on a cost recovery basis and therefore will charge the groups using the service only the arising costs! In addition the Karolinska Institutet is obliged to take VAT and overhead from non-KI customers. VAT can be reclaimed from the Swedish tax office. The administration will help you in case of questions.
Approval from ethical committees
Each research group is responsible for obtaining an approval from the local ethical committee for the transgenic experiment. Together with the application form and the respective appendix for pronucleus or blastocyst injection, the research group has to send in a copy of their ethical permission. Without this copy and the signed forms KCTT can not start the injections.
Last updated: 2010-02-19
|
Stephan Teglund, Research Associate (PhD / Department of Biosciences and Nutrition)
Experimental models
|
|
|
|
