Transgenesis Techniques: Principles and Protocols
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If the proportion of ES cell descendents in the coat of the animal is high, the probability that ES cells are represented in gametes is also high, since ES cells mix thoroughly with host cells early in embryogenesis. For the first 20 years of targeted mouse modeling, the ES cells were used almost exclusively. The ES cells used most commonly are from the strain of mice, while the host embryos are from the C57BL6 strain of mice. For much more information on the different types of ES cells, their characteristics and coat colors, please see the Generation of Chimeras page.
One of the simplest ways to study gene function in a mouse is exogenous expression of a protein in some or all tissues. For this type of genetic modification, a new piece of DNA is introduced into the mouse genome. This piece of DNA includes the structural gene of interest, a strong mouse gene promoter and enhancer to allow the gene to be expressed and vector DNA to enable the transgene to be inserted into the mouse genome. Successful integration of this DNA results in the expression of the transgene addition to the wild type, basal protein levels in the mouse.
Depending on the goal of the experiment, the transgenic mouse will exhibit over-expression of a non-mutated protein, expression of a dominant-negative form of a protein, or expression of a fluorescent-tagged protein. By definition, transgenesis is the introduction of DNA from one species into the genome of another species. Many of the first transgenic mice fit this description well as they were generated to study the overexpression of a human protein, often an oncogene 6. Currently, the phrase "transgenic mouse" generally refers to any mouse whose genome contains an inserted piece of DNA, originating from the mouse genome or from the genome of another species, and the term includes the standard transgenic mouse as well as a knockin or knockout mouse.
To generate a standard transgenic mouse, a bacterial or viral vector containing the transgene and any desired markers are injected into the pronucleus of a fertilized mouse egg.
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The DNA usually integrates into one or more loci during the first few cell divisions of preimplantation development. The number of copies of the transgenic fragment can vary from one to several hundred, arranged primarily in head-to-tail arrays, and the transgenic founder mice are mosaic for the presence of the transgene.
Founders are very likely to have germ cells with the integrated transgene, and therefore will be able to vertically transmit the integrated gene, and all cells of the progeny transgenic mouse contain the transgene. This method is relatively quick, but includes the risk that the DNA may insert itself into a critical locus, causing an unexpected, detrimental genetic mutation.
Alternatively, the transgene may insert into a locus that is subject to gene silencing. If the protein being expressed from the transgene causes toxicity, excessive overexpression from multiple insertions can be lethal to some tissues or even to the entire mouse. For these reasons, several independent lines mice containing the same transgene must be created and studied to ensure that any resulting phenotype is not due to toxic gene-dosing or to the mutations created at the site of transgene insertion.
A protocol for preparing DNA for a pronuclear injection to make transgenic mice is posted in the Methods section. Non-typical expression of a gene, usually due to a change in or replacement of the promoter of the gene.
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Can cause an expression level that is higher, lower or differently regulated for that cell type. A region of DNA that is separate from the Gene Promoter that also affects the transcription of the gene. Enhancers have been found within introns or even several kilobases from the 5' or 3' end of the gene. A regulator region of DNA a short distance from the 5' end of a gene that acts as the binding site for RNA polymerase. The chromosome complement of an individual or cell, as seen during mitotic metaphase.
In mice, a normal karyotype has 40 sister chromatid pairs. The globular mass of cells formed by the cleavage of the fertilized egg in the first stages of its development.
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- Transgenesis techniques : principles and protocols.
A gene that contributes to the production of a cancer. Oncogenes are generally mutated forms of normal cellular genes. Polymerase chain reaction- a method for amplifying specific DNA segments which exploits certain features of DNA replication. Able to develop and differentiate into any of a large number of cell types.
This second edition devotes much space to the creation of genetically modified murine strains, providing access to the germline both by conventional pronuclear injection and by retroviral and adenoviral infection. Extensive coverage is also given to the generation, maintenance, and manipulation of embryonic stem cell lineages, with protocols for both constitutive and conditional Cre-lox or the Flp-frt systems gene targeting.
Transgenesis Techniques: Principles And Protocols
Additional chapters provide cutting-edge techniques for the cryopreservation of both male and female germlines, for the generation of transgenic sheep by nuclear transfer, and for the basic analysis of transgenic organisms. Comprehensive and highly practical, this second edition of Transgenesis Techniques: Principles and Protocols updates and expands the acclaimed first edition with a wealth of easy-to-follow cutting-edge protocols and practical tips for producing transgenic animals and analyzing their integrated transgenes.
From Reviews of the First Edition " It will also remind you of the enormous potential that transgenesis has to offer the biologist. Transgenic research will underpin many future applications from evaluating viral vectors for gene therapy and vaccination to bioreactors for the production of proteins, from models of disease to screening tools for biological activity, from genome mapping to gene function. This book details protocols to let you participate in this.
Excision of genomic regions by SSR enzymes was a valuable tool to develop transgenic models that bypassed some of the limitations of the first generation of knockouts. To achieve genetic inactivation in cell type-specific manner, it is necessary that the recombinase is expressed in the cells containing specific exons or entire genes flanked by FRT or LoxP sequences.
To do so, two independent transgenic mice are crossed: one carrying the recognition sites flanking the region to be excised and the other displaying the coding sequence of the SSR enzyme under the control of cell-type-specific promoters. Pups carrying both transgenes will have knockout Cre- or Flp-expressing and non-recombined Cre- or Flpnegative cells Fig. In addition to the excision of coding regions, SRR-mediated excision was also used to generate chimeric proteins e.
Therefore, application of SSR enzymes represented a breakthrough for in vivo genetic modifications Branda and Dymecki Cre under the control of a cell type-specific promoter CPR and the other containing coding regions of the gene of interest flanked by LoxP sequences. Offspring of this mating carrying both transgenes will undergo genetic inactivation only in cells expressing Cre that will recombine the LoxP flanked region. In cells without Cre expression no recombination or gene inactivation will occur.
The Cre-ERT2 only translocates to the nucleus and catalyze the recombination after tamoxifen treatment. C Knockin recombination reporter mice usually carry a construct with a stop codon flanked by LoxP sequences followed by a reporter gene RG. In mice carrying this transgene, RG is turned on only in cells expressing Cre, where the stop codon was excised. In the Cre negative cells, the RG is not expressed. These approaches were used in mice to switch on transgenes in a tissue-specific manner Lakso et al. By the time the first SSR-dependent knockin mice was generated, it was already known that SV40 T overexpression triggered tumorigenesis in the lens Mahon et al.
Researchers aimed to establish a proof-of-principle that Cre expression could be restricted to a specific tissue and drive recombination of LoxP sites in vivo.
The offspring of these mice expressed SV40 T only in the lens and developed lens tumors, showing that SSR systems could function in vivo Lakso et al. SSR enzymes were also used to improve the procedures of transgenic mice generation.
Transgenics: CCAC Guidelines and Animal Welfare
As explained, antibiotic resistance genes ARG are required for selection of ES-containing engineered transgenes. However, it was demonstrated that, in some cases, ARG constructs could disrupt gene expression nearby the transgene locus Scacheri et al. To solve this problem, recognition sites of Cre or Flp surrounding the ARG region were inserted in the transgene. When mice containing this transgene were crossed with others expressing the correspondent SSR enzyme ubiquitously, the ARG region was excised from the genome of the offspring Ren et al.
Mouse lines containing modified versions of the Cre allowed a more refined control of the timing of recombinasemediated excision Tronche et al. The Cre-ERT2 is a chimeric protein that only translocates to the nucleus in the presence of tamoxifen. Therefore, regardless of ubiquitous or cell type-specific expression of Cre-ERT2, the Cre-mediated recombination will only occur after tamoxifen treatment, providing sophisticated timing control capabilities Figure 1 B Ahn and Joyner , Lagace et al.
Interestingly, the daughters of the recombined cell will also express the reporter gene van Amerongen et al. An extremely important requirement when using the transgenic lines that express SSR enzymes is to characterize the pattern of expression and activity of the recombinases in order to define where and when genetic inactivation will occur. Multiple approaches have been used, such as immunostaining for SSR enzymes and mouse lines in which the expression of a reporter gene is dependent on the SSR activity Buchholz et al.
In mice carrying this transgene, only cells with Cre activity will recombine the stop codon and express the reporter gene Fig.
As mentioned, these LSL cassettes were also used for Cre-dependent overexpression. In this case, usually the LSL is located in between the coding sequence of the gene and its transcription initiation site so the removal of the stop codon allows overexpression of the gene.