1. Manipulation of the mouse genome: a multiple impact resource for drug discovery and development 
Haydn Prosser, Sohaila Rastan 
Trends in Biotechnology 
Volume 21, Issue 5, Pages (May 2003)
DOI: /S (03)

2. Fig. 1
Gene addition and knockout (KO). For the creation of a gene addition transgenic the purified transgene fragment is microinjected into the male pronucleus of a fertilized egg. The transgene integrates at random, the developing embryo is transferred to the oviduct of a pseudopregnant female recipient and the resulting litter of mice is analysed by Southern blot for ‘founder’ transgenic lines. The progeny of founder animals are analysed for germline transmission of the transgene, and appropriate expression pattern and level of expression. For the creation of targeted or gene KO transgenic mice a targeting construct is introduced into pluripotent embryonic stem (ES) cells by electroporation. The most generally used targeting vector is a replacement vector, depicted here, in which a segment of genomic DNA is replaced by an exogenous piece of DNA. Basically this consists of a drug-resistance marker for positive selection (e.g. the neomycin resistance, or neo, gene driven by the pgk promoter) flanked by two arms of homology. ES cells that integrate the neo gene within their genome will give rise to colonies under drug selection. Integration can be by homologous recombination involving a double crossover event between the target chromosome and the homologous arms, or by random integration. Random integrants can be selected against by use of a negative-selection marker within the targeting vector backbone (e.g. HSV thymidine kinase, tk). ES colonies are picked and expanded for Southern analysis to identify the correctly targeted homologous recombinant clones, which are micro-injected into the cavity of day four (blastocyst) stage embryos. Blastocysts are transferred to the uteri of pseudopregnant female recipients. The ES cells will contribute to the formation of a chimeric mouse, composed of blastocyst donor and ES-cell-derived cells. Chimeras are bred with wild-type mice to assess germline transmission of the ES cell genome by coat colour and targeted mutation by Southern analysis. The N1F0 offspring are usually intercrossed to give N1F1 litters, which will contain a Mendelian distribution (1:2:1) of wild type, heterozygotes and homozygote mutant animals.
Trends in Biotechnology  , DOI: ( /S (03) )

3. Fig. 2
Inducible knockout or overexpression using the Cre/lox system. The bacteriophage P1-derived Cre recombinase acts to effect site-specific recombination between 34bp DNA recognition sequences termed loxP sites [19]. Upon Cre-mediated recombination between two loxP sites in cis, the intervening DNA sequence will be deleted. This mechanism can be employed to induce either overexpression [29–31] or knockout (KO) [21–29] of a gene. To make the Cre activity inducible the Cre gene is expressed as a fusion with a mutant form of the oestrogen receptor (CreERTM), which is able to bind the drug tamoxifen, but not naturally occurring oestrogen. The inducible Cre mouse line is crossed with mouse lines carrying the loxP sites. For overexpression the loxP sites flank a transcription termination sequence that interrupts expression of the gene being studied from a 5′ promoter. Upon injection of tamoxifen the induced Cre activity will delete the transcription termination sequence, allowing expression of the gene of interest in tissues determined by the specificity of the promoters used for both of the transgenes. For inducible knockouts the loxP sites are introduced so as to flank exon(s) (flox) of a target gene using homologous recombination such that deletion of the floxed exon(s) will result in a null mutation. Upon injection of tamoxifen into a mouse line containing Cre-ERTM and both alleles of target gene in the floxed configuration, Cre activity will cause the specific deletion in both alleles, and inducible knockout of the target gene.
Trends in Biotechnology  , DOI: ( /S (03) )

4. Fig. 3
Tetracycline-induced gene expression and gene knockout. Inducible promoter systems, such as the tet-on and tet-off systems, have proven effective as a means of regulating transcription in transgenic mice [41]. The bacterial tetracycline repressor is fused to a transcriptional activator (HSV VP16) to form a chimeric transcription factor. In the tet-off system binding of the tetracycline analogue doxycycline to the tet-repressor prevents binding to the tet operator (tetO) and thus inactivates a target gene, which is transcribed from a tetO and minimal promoter combination in the absence of doxycycline. In the tet-on system the tet repressor has been modified by mutation so that it binds to tetO upon introducing doxycycline, thus activating the transcription of a previously inactive target gene. By breeding together transgenic lines that harbour the tetO target gene (responder) line and one of the effectors (tet-on or tet-off) it is possible to express or switch-off transcription in an inducible manner. By gene targeting in embryonic stem cells and then interbreeding the resulting mouse lines to create a compound transgenic, it is possible to introduce the effector and responder components of the system into the two alleles of the target gene. The transcriptional control of the target gene will thus be subverted to incorporate a doxycycline-regulated control mechanism, in which the timing and expression levels of the target genes' expression are dependent on the timing and dosage of doxycycline administration [38].
Trends in Biotechnology  , DOI: ( /S (03) )

5. Fig. 4
Chromosomal engineering in embryonic stem (ES) cells to facilitate recovery of ethylnitrosourea (ENU) mutations. Chromosome engineering techniques (inversions and deletions) have been developed to facilitate screening protocols for ENU-induced recessive mutations [8,47]. Vector backbones containing the 5′ and 3′ halves of the Hypoxanthine Phosphoribosyltransferase (HPRT) gene mini-locus are consecutively targeted into the same chromosome within an ES cell to create endpoints for the chromosomal rearrangements. Chromosomal engineering is achieved by Cre expression and further rounds of drug selection. Depending on the orientation of the integrated loxP site, which itself is determined by the orientation of the genomic insert within the vector backbone, a chromosomal deletion or inversion (balancer chromosome) will result. Balancer chromosomes possess the important characteristic of preventing meiotic recombination with its wild-type homologue within the inverted region. The vector backbones also carry the Tyrosinase (C) and Agouti (A) genes, which together give a dominant yellow pigmentation in mice, allowing the presence of the chromosomal alteration to be traced without the necessity for genotype analysis. ENU-induced mutations are introduced to the engineered genetic background by breeding with male mice mutagenized within their germ cells. The presence of a recessive mutant phenotype will be unmasked if it occurs within the region encompassed by a chromosomal deletion. Alternatively, the use of balancer chromosomes allows the genotype within the inverted chromosomal region to be unequivocally interpreted from the coat color and phenotype of the end-point mutations, which are always linked to the inversion. Using the breeding scheme illustrated it is possible to obtain two distinctive classes of mouse, a mutant class for phenotype screens and a carrier class for maintenance of a mutant stock.
Trends in Biotechnology  , DOI: ( /S (03) )

6. Fig. 5
Gene knock-in and ‘humanization’. The figure depicts a procedure that was used to convert a mouse gene to its human orthologue in support of drug development [66]. In this example the mouse gene had a simple genomic structure, with only two exons, one of which codes for the protein. A targeting construct was engineered in which the orthologous human cDNA was flanked by 5′ and 3′ homologous arms from a mouse genomic library and a loxP-flanked pgkneo positive selection cassette. The human Kozak translation initiation sequence was retained within this construct and positioned within part of the mouse 5′ untranslated region (UTR). Successful gene targeting in embryonic stem cells inserted the human orthologue under the control of the mouse transcriptional apparatus while deleting the mouse coding region. In this case, to avoid any effects on transcription of the targeted gene, the pgkneo selection cassette was removed from mouse embryos by pronuclear injection of the Cre expression plasmid pCAG-Cre. It should be noted however, that with more complex genomic structures the effects of the knock-in procedure on RNA transcript are not always easy to predict, raising the possibility that the gene knock-in might not express correctly because of induction of aberrant alternate splicing events.
Trends in Biotechnology  , DOI: ( /S (03) )

7. Fig. 6
Techniques of mouse genetic manipulation are now impacting pharmaceutical Research and Development throughout the pipeline.
Trends in Biotechnology  , DOI: ( /S (03) )