Transgenic mice
Transgenesis has become a major tool for biologists. Although most genetically modified animals such as mice are generated for basic sutdies, physiology and pathology of farm animals such as pigs, sheep or cattle are more similar to those of human and represent more appropriate models than rodents. Transgenic famr animals have been shown to mimic human diseases.
Several companies such as GTC-Biotherapeutics are also actively pursuing “gene pharming” which entails the production of recombinant pharmaceutically active human proteins in the mammary gland or blood of transgenic animals. The use of transgenic chickens as bioreactors to synthesise therapeutic proteins, as a component of egg yolk or white, has several advantages over other systems including a shorter timescale for setup.
Random Integration of Foreign DNA into host chromosomes
In this model, there is random integration of foreign DNA into host chromosomes.
Construct Creation (1) choose a reliable promoter/enhancer proven in transgenic mice (2) include endogeous or heterologous introns (3) use a strong polyadenylation signal (4) create flanking restriction sites because transgene must be excised from plasmid for microinjection
Microinjection: (1) Microinject the DNA into male pronucleus fertilized mouse egg (0.5 dpc eggs).
Implantation: (1) implant fertilized eggs into oviduct of pseudo pregnant foster mother
Offspring: are referred to as “founders”
Gene Targeted or “knockout”
In this model, there is homologous recombination of foreign DNA into host chromosomes. These transgenic mice have been very useful in understanding how the removal of a particular gene product affects the immune system. Related techniques can be used to produce conditional mutants, in which a selected gene becomes disrupted in a specific tissue at a certain time.
The standard methodology for producing a transgneic embryo requires introducing a targeting construct, which is designed to integrate by homologous recombination with the endogenous nucleic acid sequence of the targeted gene, into a suitable ES cells. The ES cells are then cultured under conditions effective for homologous recombination between the recombinant nucleic acid sequence of the targeting construct and the genomic nucleic acid sequence of the host cell chromosome. Genetically engineered stem cell that are identified as comprising a knockout genotype which comprises the recombinant allele is introduced tinto an animals, at an embryonic stage using standard techniques such as by microinjecting the genetically enginerred ES cell into a blstocyst. The resulting chimeric blastocyst is then placed within the uterus of a psuedo pregant foster mother for the development into viable pups. The resulting viable pups include potentially chimeric founder animals whose somatic and germline tissue comprise a mixture of cells dervied from the genetically engineered ES cells and the recipient blastocyste. The contribution of the genetically altered stem cell to the germline of the resulting chimeric mice allows the altered ES cell geome which comprises the disrupted target gene to be transmitted to the progeny of these founder animals thereby facilitaitng the production of transgenic “knockout animals” whose genomes comprise a gene which has been genetically engineered to compirse a particular defect in a target gene. (Li, US2007/0028316).
The most common homolous recombination system is Cre/lox.
Cre/LoxP: is a simple two-component system currently reconbized as a powerful DNA recombination tool. Cre recombinase can catalyze the reciprocal site-specific recombination of DNA at 34-bp loxP sites. When two loxP sites are in the same orientation on a linear DNA molecule, Cre-meditated intramolecular recombination resolves with the excision of the Lox–flanking region. It does not require any host cofactor or accessory protein.
How does this work in the transgenic mouse model? The target gene is replaced by a fully functional version of the gene that is flanked by a pair of short DNA sequences, called lox sites, that are recognized by the Cre recombinase protein. Cre recombinase is a 38-kDa protein that belongs to the integrase family of site specific recombinases. It catalyzes cofactor independent recombination between two of its recognition sites, called loxP. The 34 bp consensus for loxP sites consists of an asymmetrical core spacer of 8 bp, defining the orientation of the loxPsite and two 13 bp palindromic flanking sequences. A NDA sequence that is flanked by loxP sites is excised when the loxPsites are convergently oriented, whereas the sequence is inverted when the loxP sites are divergently oriented. Cre recombinase is able to act on both inter and intramolecular loxPsites, although recombination of intramolecular lox sites is kinetically favorable.
The transgenic mice that result are phenotypically normal. They are then mated with transgenic mice that express the Cre recombinase gene under the control of an inducible promoter. In the specific cells or tissues in which Cre is switched on, Cre catalyzes recombination between the lox sequences thereby excising a target gene and eliminating its activity.
Example of generation of Act1-deficient mice: The Act1 genomic clone was obtained by screening a 129/sv BAC library. The HindIII/BamHI genomic fragment containing the exon 2 of the Act1 gene (residues 1-268, containing the first ATG) was subcloned into the Tri-Neo vector (contianing three loxP sites). The neomycin resistance gene (Neo) was inserted between the first and second loxP sites, the HindIII/BamHI genomic fragment contianing the exon 2 of the Act1 gene was flanked by the second and the third loxP sites. The resulting DNA fragment including lox-Neo-lox-exon2-lox was then subcloned into the pBlueScript vecotr. For construction of the Act1 gene targeting vector, the 5′ arm was subcloned upstream of lox-Neo-lox-exon2-lox in pBS, while the 3′ arm was subcloned dosntream of lox-Neo-lox-exon2-lox in pBS. Diphtoxin A gene was inserted downstream of the 3′ arm for negative selection. The resulting Act1 gene targeting vector was then linearized with Not I, electroporated into 129/sv ES cells, followed by selection for G418 resistant ES clones which were then transfected with Cre to remove the Neo drug marker and the exon 2 of Act1, followed by Southern blots with two probes (prb1 and prb2) located outside of each end othe of the targeting construct. Targeted ES cells (Act1-null) were injected into mouse blastocycts to generate WT, hterozygous and momozygous mice (BAL/c). Act1 deficient mice and their age and gener matched WT littermates from these intercrosses were used fo experiments. (Li, US2007/0028316).
Construct Creation (1) must use mouse genomic DNA (isogenic DNA isolated from strain 129 mouse genomic library) as opposed to transgenic model above where you can use foreign DNA. (2) use 5-10 kb of homology to favor higher frequency of recombination (3) delete or mutate critical coding sequences to insure gene inactivation. This can be achieved by inserting the selection neo gene into one of the exons of the gene of interest.
Transfection: (1) This model requires the extra step that the construct be transfected into embryonic stem (ES) cells. The ES are from the inner cell mass of a mouse blastocyte and cultured on a feeder layer of fibroblasts or in the presence of leukemia inhibitory factor (LIF). Under these conditions the ES cells grow but remain pluripotent (undifferentiated; can differentiate in a variety of directions generating distinct cellular lineages like germ cells, blood vessels, etc).
Only a few rare ES cells will have their corresponding normal genes replaced by the altered gene through a homologous recombination event. f the introduced DNA includes a selection gene, then those ES cells containing the desired gene can be selected
Microinjection: (1) After the altered ES cells are clonally expanded in cell culture, microinject the ES cells into inner cell mass of an early mouse embroyo (cavity of 3.5 dpc blastocyct)
Implantation: (1) the blastocyst is surgically implanted into 2.5 dpc pseudopregnant foster mothers
Offspring: are chimeras, meaning that they are composed of cells derived from normal cells of the host blastocyst and from the genetically altered ES cells. Initial offspring carry only a single copy of the targeted allele (+/-), but animals homozygous for the null allele (-/-) are generated by mating of 2 heterozygotes.