D Chopra, H Rehan
biomedical research, gene regulation, genetically modified, transgenic mice
D Chopra, H Rehan. Genetically manipulated mice models as research tool. The Internet Journal of Pharmacology. 2009 Volume 8 Number 1.
Genetically modified mouse models have emerged as an excellent approach to provide insight into gene regulation, development, pathogenesis, and the treatment of disease.Transgenic models are the biological tools that aid target discovery, validation, and production of therapeutic proteins. Despite several theoretical and technical challenges in generation of knockout models, transgenic technology holds great potential in many fields including medicine, industry. This short review covers briefly the methods used to produce transgenic mice and the importance of these genetically modified animals in biomedical research.
Recent progress in genetic engineering and molecular genetics permitted the development of technique for manipulation of genes in intact animals. The animals expressing exogenous gene sequences using these technologies were termed as transgenic . This term was first used by Gordon and Russel. By using transgenic technologies the gene of interest is inserted and is made to express in a new genetic locus. Ever since transgenic technology has been developed it has been used in almost every field of research. Application of animal transgenesis is used to study the role of certain genes in the initiation / progression of a disease, provide new insight into the genetic origin of disease, the pathogenesis and development of potential new therapeutic targets.
Two main transgenic procedures used to develop these animals are DNA microinjection and embryonic stem cell technique/gene targeting. Amongst these two, DNA microinjection was the first technique to be developed and was first applied to mice  followed by other species such as rats, rabbits, pigs, sheep, birds and fish. As the generation time of mice is the shortest and the mouse genome is the most assessable mammalian genome to manipulation, it is the most widely used transgenic animal. Thus the present review will focus on transgenic mice.
Methods of Production of Transgenic mice [2, 3].
This technique involves injection of a gene of interest into the pronucleus of a fertilized ovum. First step is to construct a transgene, which consists of the gene of interest, plus a promoter sequence that will control the function of the gene in the new animal. Then early embryo (one cell stage fertilized zygote) is harvested from the oviduct of pregnant donor mice. An extremely fine needle is used to inject a tiny amount of a solution containing many copies of the transgene into the male pronucleus of the fertilized egg. Injected embryos are surgically transferred into oviduct of pseudopregnant mice (generated by mating female mice with vasectomized male). The hormonal changes that will be produced in pseudopregnant mice will provide suitable environment for the survival and implantation of the transferred embryos. DNA from the progeny can be screened for the presence of transgene, the DNA, can be isolated from mouse tail biopsy. The mice, which transmit the transgene to the germ line, are referred to as founder mice. These founder mice are bred in order to produce animals that are homozygous for the transgene insertion.
The advantage of this procedure is that by using this technique rate of integration of transgene is the highest as compared to other known methods. The disadvantage of this method being that the insertion of DNA is random and there is high probability that the transgene may not insert itself into a site on the host DNA that will permit its expression.
This procedure enable site-specific introduction of mutation into the murine gene. For this embryonic stem (ES) cells are derived from mouse embryos. Most ES cell lines are derived from 129/Sv strain of mice. A targeting construct is designed to achieve homologous recombination (in which the native target sequence is replaced with the target gene). The target construct is then introduced into ES cells. Homologous recombination results in incorporation of targeting construct into the endogenous gene locus. Isolation of ES clones is done and the homologous recombinant ES cell clones are then injected into the blastocoele cavity of 3.5-day-old embryo at blastocyst. The blastocysts are reimplanted into the pseudopregnant mice. The resulting progeny will be chimeric, partly derived from the injected ES cells and partly the host embryo. Chimera mice can then be crossed with wild mice, the resulting offsprings will have 50% probability of harboring a targeted allele. Heterozygotes can then be intercrossed to generate mice homozygous for the targeted allele.
The null mutations are typically constitutive i.e, they are present throughout embryonic and postnatal developmental stages. Transgene or gene knockout may cause unexpected embryonic or neonatal lethality, by interfering with embryo development. If the aim of an animal model is to study the effect of the transgene or gene knockout on adult physiology or development of a disease, embryonic lethality limits the usefulness of such a model. Also if compensation occurs for the loss of gene product during the development then the functional significance of gene product in the adult mice will be underestimated. Thus in order to circumvent this problem the null mutation of a gene can be rendered conditional (by molecular switch). Such models are referred to as binary models.
Tetracycline control transgene expression—Tet-system.
First introduced by Bujard laboratory in 1992 and since then many refinements have been made in this system . This system is based on a tetracycline resistant operon from E.coli. This system requires the generation and crossing of two different lines of mice. The first mouse line expresses the Tet transactivator (a transcriptional factor which regulates the expression of any gene downstream of its promoter sequence) in specific tissue. The second mouse line harbors the gene of interest driven by Tet op promoter. Inducibility in Tet system depends upon the presence of tetracycline or tetracycline analog. In the Tet-off system, tetracycline or its analog inhibits the interaction between Tet transactivator and Tet op promoter. When tetracycline or its analog is present, the Tet transactivator binds it and loses its capacity to bind to Tet op, thus suppressing the transcription of the gene of interest.
Disadvantage of Tet-off system is that induction of transgene only in the adult animal requires the persistent exposure of mice to tetracycline or its analog .
Another conditional gene knockout strategy is the Cre-Lox strategy. This method is based on the enzyme Cre recombinase that is present in the bacteriophage P1. Bacteriophage P1 uses this enzyme for integration of their DNA into the genome of their bacterial host. Essential component of this process is the recognition of a unique34 bp sequence termed as LoxP site. The enzyme Cre recombinase recognizes these sites and removes the intervening segment of DNA that lies in between two copies of LoxP site. This also works in eukaryotic system. Flanking a sequence with two similarly oriented LoxP site will lead to its excision in presence of Cre recombinase. To apply this system two separate transgen lines are developed. First transgenic line is created bearing the silenced transgene with flanking LoxP site. LoxP sites are placed in intronic region where they are presumed inert, and they don’t interfere with normal gene trancription. A second transgenic line which expresses the Cre recombinase, driven by tissue specific promoter. Once transgenic mice exhibiting desired pattern of Cre expression is identified they are crossed with transgenic line bearing LoxP site resulting in generation of mice with restricted pattern of gene inactivation [2, 6, 7].
Transgenic mice are used in almost every field of research including cardiovascular, central nervous system, immunology, oncology, toxicology, carcinogenicity testing.
Transgenic mice have been used for understanding the pathology of various cardiovascular diseases like diabetes, obesity and hypertension and for developing new treatments.
The role of plasminogen activator inhibitor-1 (PAI-1) in obesity was investigated by using transgenic mice. Transgenic mice overexpressing murine PAI-1 were kept on standard food or on high-fat diet (HFD) for 15 weeks. The results showed that there was a significant reduction in fat deposits in transgenic mice kept on the HFD. Fasting insulin and total cholesterol levels on the HFD were also lower in transgenic than in wild type mice .
Transgenic mice overexpressing 11 beta hydroxysteroid dehydrogenase type 1 enzyme suggested the role of local glucocorticoid formation in visceral obesity .
The potential role of uncoupling protein 3 in treatment of obesity studied in transgenic mice by over expressing UCP3 in skeletal muscle and it was found that 18- fold increase of UCP 3 mRNA in skeletal muscle reduced diet induced obesity .
Hypertension is a complex and multifactorial disease, transgenic mice models have proved to be useful to study pathogenesis of and to find new therapy for its treatment.
The role of matrix metalloproteinase (MMP-9) in the progression of hypertensive vascular disease in vivo was evaluated using transgenic mice in which MMP-9 gene was knockout. The study showed that absence of MMP-9 activity results in vessel stiffness and increased pulse pressure .
Role of thyrotropin releasing hormone (TRH) in diabetes was studied using transgenic mice and it was found that TRH knockout mice were hyperglycemic. This finding suggested that TRH might have a physiologic role in the regulation of carbohydrate metabolism and in the treatment of diabetes .
Central nervous system
Transgenic mice lacking hypothalmic neuropeptide orexin displayed frequent episode of inactivity characterized by sudden collapse of head on EEG. These episodes were found to be similar to narcoleptic attacks observed in humans. Thus this mouse model revealed novel role of orexin in sleep regulation and represent an important model for examining the pathophysiology and treatment of narcolepsy .
No animal model of alzheimer’s disease existed before the transgenic animals were developed. The transgenic model developed for this disease resembles much of the human pathology and is used in the search of novel therapeutic approach against this disease.
Schenk et al studied the role of A 42 in the disease progression in APP (amyloidal precursor protein) transgenic mice. The study showed immunization of APP transgenic mice with protein A 42 before the disease is established resulted in disease prevention and if immunization in older animals, the disease progression is inhibited. Thus the study suggests that vaccination with A 42 protein could have potential as a therapeutic approach against alzheimer’s disease. Over expression of local hippocampal IL 1 in an alzheimer’s disease transgenic mouse model resulted in plaque amelioration. This showed the beneficial role of IL 1 in alzheimer’s disease and may be a potential therapeutic approach and may be a potential therapeutic approach to protect against alzheimer’s disease .
Huntington ’s disease
Transgenic mice have been used to test new therapeutic approach for Huntington ’s disease. Huntington ’s transgenic mice were crossed to animals transgenic for a dominant negative caspase-1 transgene. This demonstrated that inhibition of caspase could delay onset of symptoms and prolong survival in the transgenic mice .
The role of adenosine A1 receptor in neuroprotection during epileptogenesis has been studied using transgenic mice in which the adenosine A1 receptor was knocked out. The study showed kainic acid injection in A1R KO mice displayed massive neuronal cell loss in the ipsilateral hippocampus and the contralateral hippocampus was also significantly affected. The study concluded that adenosine A1 receptor is crucial in keeping epileptic foci localized and open up a new dimension of A1 receptor’s role in preventing the progression of epilepsy . Role of GABA b R1a subtype in etiology of atypical epilepsy was tested using transgenic mice overexpressing GABA b receptor R1a subtype. The results showed that transgenic mice develop spontaneous, recurrent bilaterally synchronous 3-6 Hz slow spike and wave discharge between 2-4 months of age, which were blocked by ethosuximide and exacerbated by baclofen .
Transgenic mice have been very useful in understanding the function of oncogenes and tumour suppressor genes, mechanisms behind metastasis. Model of multistage tumour progression based on transgenic expression of SV 40 large T antigen in beta cells of the pancreas (rip1 tag2 model) was used to study the role of adhesion molecules in metastasis. Transgenic mice expressing dominant negative E- cadherin gene on Rip1 Tag2 transgenic background showed the essential role of E- cadherin in tumour progression. Similarly the role of NCAM was also demonstrated [18,19].
Understanding the mechanisms behind metastasis can provide valuable therapeutic approach.
The role of adenosine A2A receptor in physical dependence and place conditioning induced by 9- tetrahydrocannabinol (THC) was studied in transgenic mice lacking A2A receptor. This showed behavioral response induced by acute THC remained unaffected in mutant mice. Tolerance to chronic administration was also similar in both wild and mutant mice. But the somatic manifestations in response to withdrawal of THC were attenuated in mutant mice. Also a significant reduction of THC induced rewarding and aversive effects was found in mutant mice as compared to wild type. Thus for the first time specific involvement of A2A receptors in addictive properties of cannabinoids was revealed . Specific role of A2A adenosine receptor in the behavioral responses induced by nicotine was investigated by using transgenic mice lacking A2A adenosine receptors. Acute administration of nicotine showed a similar decrease of locomotor activity in mutant mice and wild type. Also anti nociceptive responses elicited by nicotine were unaffected in mutant mice. Nicotine induced conditioned place preference was suppressed in transgenic mice model which suggested that A2A adenosine receptor is an important factor that contributes to rewarding properties of nicotine .
Beneficial role of IL-6 against ethanol induced oxidative stress was determined by using IL-6 deficient mice. Ethanol consumption resulted in stronger induction of malonyldialdehyde in transgenic mice lacking IL-6 as compared to wild type mice. IL-6 administration in vitro prevents ethanol mediated induction of reactive oxygen species. In conclusion IL-6 protects against ethanol induced oxidative stress and thus alcoholic liver disease .
Role of cytochrome P450 1A2, cytochrome P450 2E1 in acetaminophen toxicity was established when it was seen double knockout mice lacking both cytochrome P450 1A2, cytochrome P450 2E1 were significantly more resistant to acetaminophen toxicity than either of the single knockout lines .
Cytochrome P450 1A1 plays an important role in benzopyrene toxicity was established by deleting cytochrome P450 1A1 from mouse genome. The cytochrome P450 1A1 knockout mice were found to be resistant to toxic effects of benzopyrene .
Transgenic model serve to improve the identification of carcinogen by providing gene specific mechanistic data, minimizing influence of spontaneously occurring tumours in animal species and reducing the time cost and animal usage involved. In addition to their utility in shorter term in-vivo assays for carcinogenicity, the transgenic mice models are useful for research into the mechanisms underlying carcinogenesis. Four transgenic mouse models: the TSPp53+/-, the Tg AC, the Hras 2 and the XPA-/- has ben evaluated. Each model has its own advantages and limitations. ICH (1997) in its pharmaceutical testing guideline has incorporated their use in place of long-term mouse bioassay when the traditional rat study has already been performed. The Tg AC and p53 are two most widely used models .
Tg AC model
In this model four copies of v-Has- ras oncogene fused to fetal zeta globin gene promoter are inserted. Topical application of carcinogen produces pedunculated tumours arising from epithelium. Animals are exposed to the carcinogen for 20 weeks and observed for additional 6 weeks. This model is good for detecting both mutagenic and non-mutagenic carcinogens.
Tg Hras 2 model
In this model the mice carries 5-6 copies of human H-ras protooncogene in tandem surrounded by their own promoter and enhancer region. Study duration of 6 months is sufficient to obtain positive result.
p53+/- mouse model
This model carries hemizygous knockout of p53 gene. p53+/- mice are found to be sensitive to genotoxic carcinogens and are insensitive to non- genotoxic carcinogen
XPA-/- mouse model
This model was created by deleting both the alleles of XPA gene. The protein coded by this gene is essential for detection and repair of DNA damage. This model thus has low activity of the nucleotide excision repair pathway. XPA-/- mice are found to be sensitive to genotoxic carcinogens. The tumour appears within 9 months of exposure to carcinogen.
Transgenic technology is a powerful research tool, which can save researchers time and money. Though still, in infancy this technology is finding increasing uses in the pharmaceutical industry. Its success will ultimately depend on how widely it will be incorporated into the industry.