File Name: transgenic and knockout animals .zip
Understanding appropriate nomenclature is essential due to the complexity of strain names for substrains, transgenics, knockouts, etc. To enable broader awareness of nomenclature, The Jackson Laboratory has provided the resources below:.
Genome modification technologies are powerful tools for molecular biology and related areas. Advances in animal transgenesis and genome editing technologies during the past three decades allowed systematic interrogation of gene function that can help model how the genome influences cellular physiology.
Genetic engineering via homologous recombination HR has been the standard method to modify genomic sequences. Here, we present a brief historical perspective of genome modification methods, focusing on transgenic mice models. Moreover, we describe how new techniques were discovered and improved, present the paradigm shifts and discuss their limitations and applications for biomedical research as well as possible future directions. A universal question in biology is how the genome translates into phenotypes giving rise to the endless forms of nature.
It dates back to the first evidences that genes encode individual proteins and the seminal discovery of DNA's threedimensional structure Beadle and Tatum , Watson and Crick Since then, molecular biology has developed at a rapid pace guided by the Central Dogma Crick and somehow impregnated by genetic determinism.
Over the second half of the 20 th century, advances in the generation of transgenic organisms made its way from prokaryotic to vertebrate model organisms, including mice. Integration of a foreign DNA sequences into host genomes characterized the first attempts to study gene function in vivo Brinster et al. In mice, pioneering studies generated non-targeted genetic modifications in somatic cells through microinjection of exogenous DNA into fertilized eggs Brinster et al.
Shortly after, a series of groundbreaking studies described the disruption of endogenous gene expression through targeted modifications that were transmitted through germ line cells reviewed in Capecchi The discoveries that allowed targeted genetic inactivation in the mouse genome, through the use of embryonic stem cells, granted the Nobel Prize award in Physiology and Medicine to Drs.
The establishment of this technique led to a revolution in the ability to interrogate the relevance of specific genes revealing the molecular basis of numerous biological phenomena from animal diversity to human diseases. Advances in DNA sequencing technologies allowed the study of eukaryote's genomes at unprecedented resolution and taught us that the variations found in the coding regions were too small to account for the substantial organismal differences King and Wilson , Whitehead and Sackstein , McGinnis and Krumlauf , Adams et al.
The lack of correlation between genome size or the number of protein-coding genes with organism complexity was puzzling. Additional findings supported the notion that organismal complexity arose from more elaborate regulatory networks rather than the appearance of new genes Levine and Tjian This scenario exacerbated the need to go beyond sequence comparisons and made clear the need to study gene function in various contexts e. Studies on gene targeting and genome edition developed over the last thirty years advanced transgenesis in an unimaginable scale.
Nowadays, we have learned to manipulate the genome in more efficient e. Here, we review and discuss the various methods to create targeted modifications in the genome and present a historical perspective of the generation of transgenic mice models that benefited from these advances. In the early 80's, a series of pioneer studies generated transgenic mice by microinjecting fertilized egg cells even before the establishment of homology based methods Gordon et al.
These studies performed the implantation of eggs previously injected with plasmids encoding viral antigens into pseudo pregnant recipient females. The insertion of various copies of the transgene in the genome, its expression in different somatic cell types and the transmission through germ line cells was observed. However, integration occurred in a non-targeted manner and without copy number control Brinster et al.
Using similar methodologies of zygote microinjection, transgenic rabbits, pigs and sheep were also produced Hammer et al. These studies represented a major advance in the areas of animal science and transgenic animal generation. The introduction of transgenes in the genome in a targeted manner was only possible with a better understanding of DNA repair by homologous recombination HR Folger et al.
Using this knowledge the groups led by Mario Capecchi and Raju Kucherlapati generated targeted genetic modifications in cultured mammalian cells reviewed in Capecchi , By electroporation or microinjection, exogenous DNA constructs could be introduced as a template of the target genomic sequence. These targeting vectors should have high homology with the targeted genomic sequence to be recognized by the HR machinery in order to introduce different types of modifications, including insertion or deletion.
However, the targeting event occurred in a small percentage of the transfected mammalian cells making it unfeasible to efficiently target a fertilized egg and generate a whole transgenic animal Capecchi , Capecchi Establishment of embryonic stem ES cells culture Martin allowed the use of HR-dependent modifications described above to target specific loci of mouse pluripotent cells. Few successful strategies previously used in other mammalian cell types were employed to select the ES clones that underwent HR.
Targeted ES cells carrying exogenous DNA sequences antibiotic resistance genes - ARG , such as neomycin-resistance gene neor , could be selected positive selection. However, positive and negative strategies of selection became necessary, since the integration of a targeting vector and the neor at random sites through non-homologous recombination also occurred. The use of a vector combining the herpes virus thymidine kinase gene HSV-tk , outside of the region to be recombined, with the neor allowed the selection of ES cells that contained the desired targeting.
In , the groups of Drs. As written by Dr. Capecchi, the protocol described in these papers "should be useful for targeting mutations into any gene" Doetschman et al. Following the established methodology of ES cells targeted modification, several groups developed knockout mice for various genes. In order to target desired genes in vivo , transgenic clones of ES cells were injected in the inner mass of blastocysts that were subsequently implanted into pseudo pregnant females which gave birth to chimeric animals.
Mice with different coat patterns were used to screen the offspring chimeras containing cells derived from the transfected ES cells. The chimeric animals containing genetically modified germ line cells transmitted the transgenes to their offspring. Then, heterozygous mice crossing led to the generation of homozygous transgenic mice. Approaches to inactivate an endogenous gene included the replacement of the targeted region or the insertion of a neor within the coding region to disrupt the open reading frame ORF and gene expression.
Altogether, the advances described above culminated in the generation of the first gene knockout mice Joyner et al. These pioneering methodologies also allowed the development of knockins with or without cell specific promoters, which could drive transgene expression in a restricted subset of cells or in the whole animal Okabe et al. Transgenic mice generated through HR standard methods already enabled control of gene expression timing and reversibility by the use of drug-inducible transgenes.
Two types of engineered tetracyclin receptors that work as drugmodulated transcription factors have been developed: the rTA is transcriptionally active after doxycycline treatment, while the rtTA receptor is prevented from binding DNA. To achieve drug-controlled transgene up- or down-regulation, two different lines are required: one with the expression of the receptor rTA or rtTA and the other with a transgene under the control of a promoter region responsive to the tetracyclin receptor.
In mice containing both transgenes, gene expression is regulated by the tetracyclin treatment Furth et al. Relevant findings in various areas of biomedical research e. As described below, new biological tools were developed to bypass these issues. In vivo germ line gene targeting can lead to premature death, making it impossible to evaluate phenotypes afterwards. Altogether, these negative features of transgenic organisms that carried genetic modification in all somatic cells increased the desire for alternative transgenic models with a tissue- or developmental stage- specific inactivation of the genes of interest.
Site-specific recombinases SSR have been used to modify the genome with temporal and cell type specificity. Different types of recombinases have been described, but here we will focus on the two SSR systems most widely used in transgenic models: the Cre-LoxP and Flp-FRT, members of the integrase family of recombinases Turan and Bode The CreLoxP system was first characterized in the bacteriophage P1 and is responsible for genomic recombination during bacterial division.
The Cre cyclization recombination recombinase is a 38 kDa protein that can catalyze the recombination of two specific LoxP sequences locus of crossing-over of P1 Sauer and Henderson The Flp recombinase, characterized in S.
Although not identical, the sites recognized by Cre and Flp display high similarities. These sequences are formed by 34 bp consensus sequence with two 13 bp palindromic sequences separated by an 8 bp spacer region that is responsible for the sequences orientation Sternberg and Hamilton , McLeod et al.
To enable recombination, two SSR enzymes monomers bind to each recognition site and mediate a Holliday junction between them before completing recombination.
Both systems exhibit high specificity and do not need cofactors. The orientation of the recognition site determines the genetic modification catalyzed by SSR enzymes. When the sites have the same orientation the recombinase excise the sequence in between them irreversibly, while, in the case of opposite orientation, the enzyme drives the inversion of the flanked region Grindley et al.
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. 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.
Even though the generation of transgenic animals using SSR based transgenesis represented a major advance in the field, there are several limitations.
Following publication of the sequence and analysis of a mouse strain in December ANCHOR the mouse became the animal model of choice for most laboratory experiments. The potential of mice for genetic manipulation means that their use is now often favored over rats and other rodents. The mouse makes an excellent model for human disease because the organisation of their DNA and way their genes are expressed is very similar to humans. Their reproductive and nervous systems are like those of humans, and they suffer from many of the same diseases such as cancer, diabetes and even anxiety. Manipulating their genes can lead them to develop other diseases that do not naturally affect them, and as a result research on mice has helped understanding of both human physiology and the causes of disease. Before genetic technology, mice were inbred to produce laboratory strains with particular characteristics. These inbred strains are very genetically similar, which makes them ideal for studying changes due to genetic modification.
A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to the gene itself, the DNA usually includes other sequences to enable it to be incorporated into the DNA of the host and to be expressed correctly by the cells of the host. Transgenic sheep and goats have been produced that express foreign proteins in their milk. Transgenic chickens are now able to synthesize human proteins in the "white" of their eggs. These animals should eventually prove to be valuable sources of proteins for human therapy.
Transgenic and knockout mouse models are particularly useful for studies of complex neurobiological problems. The primary aims of this.
Genome modification technologies are powerful tools for molecular biology and related areas. Advances in animal transgenesis and genome editing technologies during the past three decades allowed systematic interrogation of gene function that can help model how the genome influences cellular physiology. Genetic engineering via homologous recombination HR has been the standard method to modify genomic sequences. Here, we present a brief historical perspective of genome modification methods, focusing on transgenic mice models.
We relate the experimental designs, the pitfalls and challenges encountered, and the eventual success in developing distinctive mouse models of cancer, wherein tumors arose heritably in various organs. These early oncomice have produced a wealth of new knowledge, become topics of intellectual property, and spawned a vibrant field of cancer research that is revealing mechanisms of tumorigenesis and suggesting new therapeutic strategies for treating the human disease. Concurrently, there was considerable excitement in cancer research, with the continuing discoveries and molecular cloning of viral and then cellular oncogenes. These genes were causally implicated in particular natural cancers and demonstrably capable of inducing transformation of cultured cells that would form tumors when transplanted in appropriate host animals.
Cancer Models View all 14 Articles. The use of existing mouse models in cancer research is of utmost importance as they aim to explore the casual link between candidate cancer genes and carcinogenesis as well as to provide models to develop and test new therapies. However, faster progress in translating mouse cancer model research into the clinic has been hampered due to the limitations of these models to better reflect the complexities of human tumors.
Steps in gene targeting. ES cells indicates embryonic stem cells; neor , bacterial neomycin resistance gene; and HSV-tK , herpes simplex virus thymidine kinase gene.
The Transgenic and Stem Cells Service Center was established in and since that time, it has generated over new transgenic, knock-out and knock-in mouse animal models for investigators from UTHealth, as well as for scientists from numerous other academic institutions. These cell lines are routinely used in the Core Facility and are also commercially distributed. UT Rates. Eva M.
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