Transgenic Model

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 most common of these systems 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 requie 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 loxP sites, 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.

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.