1) DNA microinjection into single cell embryos to produce transgenic mice containing one or more copies of randomly integrated expression vector DNA.

2) Use of homologous recombination at defined genomic loci to create gene knockouts (KOs) or replacements.

Generating transgenic mice using fertilized egg cells

Purified experimental DNA is directly injected into the male pronucleus of a fertilized mouse egg using a microscope-mounted manipulator and microneedle. The three essential instruments for embryo injection are an inverted microscope with stage magnification of 400X, a micromanipulator-mounted negative pressure holding pipette (left) and a micromanipulator-mounted injection pipette (right).

The male pronucleus of a fertilized egg is injected with picoliter amounts of purified DNA (1ug/ml) by stabilizing the egg using the holding pipette. Success depends on the use of properly timed fertilized eggs and accurate injection of the male pronucleus.

Fertilized one-cell eggs for DNA microinjection are obtained from superovulating female mice that have been mated to a stud male. Following microinjection, 20-30 eggs are implanted into the oviduct of a pseudopregnant surrogate female (recently mated to a vasectomized male) leading to the production of ~5-8 live pups 19 days later. Transgenic littermates are identified by PCR analysis of tail samples performed at three weeks of age.

Mice that score positive by PCR analysis are backcrossed to non-transgenic mates to identify founder animals containing germline DNA integration that results in a Mendelian inheritance pattern of the transgene.

Cell-specific promoters can be used to direct expression of a foreign gene to tissue subtypes in transgenic mice. An experimental flow scheme is shown to illustrate how a prostate-specific promoter could be used to generate a transgenic mouse model of prostate cancer.

Northern blot analysis is needed to identify suitable founder mice. Characterization of prostate tissue in the offspring of founder mice would be done to determine how well the transgenic model mimics human prostate cancer.

What would explain the lack of gene expression of an integrated transgene in a founder mouse that tested positive by DNA analysis through several generations?


What could account for high levels of prostate-targeted transgene expression in kidney cells as observed in the P01 founder mouse in the example above (figure 8.7)?


How could you use a cell-specific transgene construct to investigate activation of a liver-specific promoter during mouse embryonic development, i.e, what would the transgene construct consist of?

Gene knockouts by homologous recombination in ES cells

Two methodological breakthroughs were required before transgenic "knockout" (KO) mice could be created that contained germline insertional loss-of-function mutations.

1) Procedures to cultivate pluripotent mouse embryonic stem (ES) cells that could be combined with non-transgenic mouse blastocysts to produce a chimeric embryo, and

2) The development of DNA cloning vectors and cell line screening strategies that allow the identification of transfected cells that have undergone a homologous recombination event following transfection with experimental DNA.

A pluripotent ES cell line is derived from the inner cell mass (ICM) of blastocyst embryos obtained from the uterus of a female mouse 3 days post-fertilization. ES cells are cultured on plates containing non-dividing embryonic fibroblasts called "feeder cells".

Experimental DNA is stably transfected into ES cells by electroporation or lipofection using vectors containing a selectable marker. PCR screening strategies and Southern Blotting are used to identify clonal ES sublines containing targeted gene knockouts.

Differences in the coat color of ES donor mice and blastocysts host mice are used to identify transgenic founder animals. The offspring have a mosaic coat color reflecting their chimeric genotype. Transgenic founder lines are identified as agouti offspring that arise from crossing chimeric and albino mice.

Improvements in the targeting vectors and screening strategies used to create transgenic KO mice have led to two basic strategies.

1) A gene insertion process that requires a cross-over event between two ends of the targeting vector and the genomic sequence.

2) A gene replacement strategy using vectors that contain two selectable markers that permit detection of double-cross over events in the target gene.

Gene insertion leads to disruption of the genomic gene copy due to a duplication of the targeting sequences and incorporation of the neor coding sequence.

Gene replacement vectors contain both positive (neo-resistance ) and negative (HSV tk) marker genes that are used to identify drug-resistant ES cells containing a predicted double cross-over event in the target locus. Ganciclovir (GC) is a suicide nucleotide analog that is phosphorylated by the HSV tk gene product, but is not a substrate for mammalian thymidine kinases.

The frequency of homologous recombination is ~1%, based on comparing the number of G418-resistant colonies to the number of G418- and ganciclovir-resistant colonies. This corresponds to an overall homologous recombination frequency of ~10^-5, when considering the total number of ES cells transfected.

Factors influencing the frequency of homologous recombination are;

1) the length and arrangement of homologous sequences in the targeting construct

2) the extent of homology between genomic sequences contained in the targeting vector and the target gene

3) the chromosomal location of the gene target.

Transgenic mouse models of human diseases

Mice have 19 autosomal chromosomes and one sex-linked chromosome, many of which contain large segments of DNA that are highly conserved between mouse and humans. One of the goals of mouse transgenesis, is to use molecular genetic approaches to create better mouse models of human diseases that are based on known genetic lesions.

A good place to start is the National Center for Biotechnology Information (NCBI) genomic biology page.

Examples of mouse/human Homology Maps showing where regions of mouse (black) chromosomes 6-10 line up with portions of human (colored) chromosomes.

Extensive lists of transgenic mouse models are available from Jackson Labs in Bar Harbor, Maine.

Table 8.1. Examples of transgenic mouse models of human diseases available from Jackson Labs.

What attribute of the agouti coat color mutation facilitates its use as a marker in mouse transgenesis using ES cell technology, i.e., is agouti a loss or gain of function mutation in the parental mouse blastocyts?


Design a PCR primer pair that could be used to identify homologous recombination of a gene replacement vector containing the neo-resistance gene.


What is the most likely explanation of no overt phenotype in a homozygous mouse KO, i.e., the KO mouse is normal despite loss of expression of the targeted gene?


How is it possible to maintain a KO strain of mice in which the gene disruption is lethal?

What is the advantage of the Cre-lox system for generating KO mice over the standard gene replacement strategy?


Why doesn't the Cre recombinase protein cause DNA damage in mouse tissues in which it is constitutively expressed, e.g., in the brain of CaMKII-Cre founder mice as described in the lab practicum?


What is the significance of the partial phenotype in the heterozygous mouse with regard to the type of genetic mutation that has been created?


How important is quantitating the eating behavior assay to understanding the role of bpl in behavior?


What might it mean if the bpl KO mouse displayed only a partial reduction in weight with only a minimal difference in eating behavior?

Creating point mutations by recombination events using Cre recombinase

A modified version of Cre-lox recombination was used by Gunther Schutz and colleagues to make a transgenic mouse containing a single mutation in the DNA binding domain of the glucocorticoid receptor.

This strategy used standard homologous recombination to put the point mutation into the GR locus by gene replacement (positive selection for neo) and screening by Southern blots. Nno outside negative selection gene was included, but newer vectors contain diptheria toxin gene to select for the double cross-over event.

In this strategy shown here, they included a step using transiently transfected a Cre recombinase expression plasmid into the KO ES cell subclone, along with negative selection for tk using ganciclovir, to select for clones that had deleted the tk-neo selection genes as a result of Cre-lox functions. This results in a recombination event that leaves the GR locus with a single point mutation. This is different than the original knock-in experiments which required two homologous recombination steps.

The point mutation introduced a unique restriction site (BsrGI) that permitted genotyping of the dim mutation .

Background

A researcher in the basic science unit of a Cardiovascular Research Center recently identified a novel calcium-activated potassium channel as a possible contributor to cerebral chronic high blood pressure and migrane headaches. The clue came from human genetic studies of an isolated population of island-dwelling people that had suffered for generations from chronic cerebral high blood pressure and migrane headaches. Standard gene mapping using SNP analysis of DNA samples from living inviduals, and from samples taken from the remains of their deceased relatives, led the investigator to identify a previously uncharacterized human gene on chromosome 11 as the candidate disease gene. He tentativley named the gene the Arterial Migraine Grinder (AMG) gene. Using mouse-human mapping data and genomic library screening methods, he isolated a 10 kb region of genomic DNA corresponding to the mouse AMG gene. The researcher hypothesized that the mouse AMG gene is required for maintaining basal blood pressure levels and that inactivation of the AMG gene in KO mice would lead to elevated cerebral blood pressure and mouse migranes.

Research Strategy
To directly test his hypothesis, the researcher cloned portions of the mouse AMG gene into a Gene Replacement vector for use in a homologous recombination knockout (KO) strategy to inactivate the gene. The pKO-AMG plasmid vector contained the bacterial beta-galactosidase gene which he used to create an AMG-LacZ gene as an in-frame fusion to exon 2 sequences. Following standard gene replacement methods, he generated a founder mouse strain from brown agouti ES cells and bred the mice to homozyogosity. Southern blot analysis and in situ cytochemistry was used to confirm that this mouse did not express the normal AMG protein, and moreover, that Lac Z enzyme activity could be detected in cerebral arterial cells in the same pattern as he had observed using anti-AMG antibodies (data not shown). With these mice in hand, he set out to collect physiological data to support or refute his primary hypothesis.