The construction of transgenic and gene knockout/knockin mouse models of human disease

Abstract

The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual's gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care.

Figure 1. Overview of the creation of transgenic mice

The fundamental strategy for the production of these strains of mice is ultimately straight-forward: the physical introduction of DNA into a pronucleus of a fertilized egg where it randomly integrates into a single chromosomal location within the genome. Injected eggs are subsequently returned to a surrogate mom to complete gestation with 2% of the injected eggs surviving the process and producing mice carrying the gene of interest (i.e., the transgene).

Figure 2. Production of transgenic mice leading to lung-specific gene expression

(A) Transgenic founder mice constitutively expressing a gene of interest specifically in the lung. In this example, transgenic founder mice expressing the gene of interest in Alveolar Type II cells are generated by the injection of an expression cassette utilizing a promoter derived from the gene encoding the surfactant protein C (pSPC) together with the gene of interest and an additional DNA fragment derived from the human growth hormone gene (hGH) that supplies required RNA processing motifs for transgenic gene expression, including exon-intron junctions and a poly A addition signal sequence (p(A)⁺). Incorporation of this transgene leads to transgenic mice constitutively expressing the gene of interest from Alveolar Type II cells of the lung. (B) Transgenic founder mice whose transgene expression is an inducible consequence of the administration of antibiotics (i.e., doxycycline and/or tetracyclin). In the example shown here, three constructs are co-injected providing the necessary components to generate mice whose transgene expression is stringently regulated. In Alveolar Type II cells, the surfactant protein C promoter (pSPC) drives expression of both an inhibitory bacterial transcription factor (tet-TS) and an activating/permissive transcription factor (tet-TAM2), each of which is capable of binding a DNA regulatory region known as tetO. In the absence of doxycycline/tetracycline the binding of the inhibitory factor (tet-TS) to the tetO region is greater than the activating/permissive factor (tet-TAM2), silencing gene expression. However, administration of doxycycline/tetracycline to mice leads to conformational changes in the transcription factors such that the affinity of the activating/permissive factor (tet-TAM2) to the tetO region is now greater than the inhibitory factor (tet-TS), inducing expression of the gene of interest.

Figure 3. Production of knockout mice: Gene targeting leading to the generation of animals deficient of specific genes of interest

Gene knockout mice are generated in a two staged process that utilizes pluripotent embryonic stem (ES) cells as a vehicle with which to translate experimental genetic manipulations into Mendelian inheritable traits in mice. (A) Using now standard “off-the-shelf” technologies a DNA targeting construct containing specific regions of homology to the gene of interest is transfected into ES cells (usually by electroporation). In the nuclei of these transfected ES cells homologous recombination events occur leading to a 1-to-1 replacement of the endogenous gene with sequence derived from the targeting construct. This process is greatly facilitated by a selection process that identifies ES cell clones that have successfully undergone a targeting event by positive (expression of a neomycin resistance (NEO^R) gene) and negative (expression of the herpes simplex thymidine kinase (TK) gene) drug selection strategies while the cells are in culture. Nonetheless, the frequency of occurrence of these homologous recombination events is incredibly small. For example, the frequency of stable transfection is <0.1% and successful homologous recombination events generally occur at a frequency of 1-5% among cell clones identified by the drug selection schemes, yielding an overall frequency of success < 1 in 10⁵ events. (B) Targeted ES cell clones containing the appropriate genetic changes that ablate expression of the gene of interest are transferred to the blastocoel cavities of 3.5 day blastocyst embryos (generally 10-15 ES cells/host embryo) and, in turn, the embryos are transferred to surrogate mothers where gestation is completed. At a frequency highly dependent on the quality and care with which the ES cells are maintained, transferred targeted ES cells contribute to embryonic development, generating ES cell-derived chimeric founder mice with varying degrees of chimerism - in this example, judged by ES cell (black strain of mice) contribution to coat color when transferred into blastocyst embryos from a white strain of mice. Commonly, one or more high chimeric founders are crossed with mice of the background strain from which the ES cells were derived to generate ES cell-derived founder mice which have inherited the targeted gene, generating a null-allele at the chosen genetic locus.

Figure 4. Development of conditional (inducible) knock out mice with temporal, cell, and/or tissue specific ablation of gene expression

To restrict the loss of gene expression in knockout mice in time or location with the body, a binary system of unique strains of mice are developed that utilizes both ES cell-derived genetic manipulations and traditional transgenic technologies. One strain is developed (LOX P) through ES cell manipulations leading to a homologous recombination event that inserts small repetitive sequence elements which are the recognition sequences of DNA recombination enzymes. One set of these repetitive sequence elements (FRT) flank the drug selection markers, facilitating the removal of these markers by breeding the ES cell-derived mouse with a transgenic animal expressing the DNA recombinase recognizing the FRT sequence elements (flippase (FLP)) either ubiquitously (i.e., in all cells) or in cells of the germline. Another set of repetitive sequence elements (LOX P) flank an important sequence region of a gene necessary for expression, including, but not limited to, protein encoding exons, RNA processing motifs, or cis-regulatory elements. In this example, the LOX P elements flank exon 3 of Gene X. The second strain of mice (CRE) necessary for this binary system is a transgenic line expressing the DNA recombinase that recognizes the LOX P repetitive elements (i.e., Cre recombinase) in a temporal and/or spatially restricted pattern. In this example, a constitutive transgenic line of mice is established expressing the Cre recombinase only in the paws of the front limbs of the animals using a “front paw-specific promoter”. When the binary system is complete and the LOX P mouse is crossed with the CRE line of mice the resulting offspring (CRE-LOX) will express Cre recombinase in the front paws and in doing so mediate the deletion of exon 3 of Gene X through a Cre-mediated DNA recombination event between the LOX P sequence elements. Thus, in CRE-LOX mice, Gene X is expressed normally in all tissues of the body with the exception of the front paws where the induced genetic ablation of exon 3 renders a null allele of Gene X in this region. It is noteworthy that in addition to spatially restricted knockouts, gene ablation can be restricted temporally through the selective generation of a transgenic Cre recombinase mouse using a promoter that mediates Cre expression at a unique time (e.g., at a specific point during gestation) or under inducible control (e.g., Ert-Cre).

Figure 5. Production of knock-in mice: The generation of animals expressing unique genetic alleles of a given gene of interest

Gene knock-in mice similar to their earlier knockout counterparts are generated in a two staged process that utilizes pluripotent embryonic stem (ES) cells as a vehicle with which to translate experimental genetic manipulations into Mendelian inheritable traits in mice. In these strategies, the DNA targeting construct not only contains specific regions of gene homology but also has a uniquely engineered mutation or sequence change such that the 1-to-1 replacement of the endogenous gene with sequence derived from the targeting construct following transfection into ES cells yields an allele in the genome of these cells containing this new sequence variant. In the example here, exon 2 is replaced with a modified version of this sequence information through this gene knock-in process. As noted earlier, targeted ES cell clones containing the appropriate genetic changes are transferred to the blastocoel cavities of 3.5 day blastocyst embryos. In turn, the embryos are transferred to surrogate mothers where gestation is completed generating ES cell-derived founder mice which have inherited the new sequence variant (i.e., the knock-in mutation), generating a gain-of-function allele at this chosen genetic locus.

Figure 6. Compound transgenic/conditional knockin mouse model of human pancreatic cancer

The complexity of neoplastic transformation occurring in pancreatic cancer was replicated in a mouse model through a unique binary approach breeding of a knockin animal capable of inducible expression of a constitutively active oncogene with a cell-specific Cre-expressing transgenic mouse. The first component of this compound model is a knockin line of mice (KRAS/Knock-In) generated using ES cells. These mice harbor an inducible allele of KRAS that was constitutively active. Specifically, the engineered KRAS locus in these knockin mice was created by inserting a genetic element upstream of a exon 1 that inhibit transcription/translation flanked by functional LoxP sites (LoxP-STOP-LoxP sequence element). In addition, the open reading frame of this KRAS allele was modified with a G to A transition in codon 12 of the open reading frame to produce a constitutively active variate of KRAS (KrasG12D) commonly found in human pancreatic ductal adenocarcinoma. The second engineered mouse of this compound model is a transgenic line (PDX-Cre) expressing the Cre recombinase exclusively in pancreatic islet cells through the use of a promoter fragment from the PDX-1 gene. Each of the component line of mice are maintained as “stand alone” strains with neither developing pancreatic cancer due to the expression of only wild type KRAS. However, offspring resulting from a cross of these mice (Pdx1-Cre^Tg/−/KRAS^G12D/+) recapitulate the full spectrum of human pancreatic intraepithelial neoplasias (PanINs); some of these lesions even becoming invasive and metastatic.

Figure 7. The utility of genetically-modified mouse models in biomedical research

Gene transfer technologies have been, and continue to be, very productive components of a larger approach with which to generate clinically relevant mouse models of human disease that are integral to our understanding of health and the development of new/novel therapeutic modalities.