FAQ

1.      What is a knockout mouse?

2.      What is a humanized mouse?

3.      What is a Pronuclear Injection?

4.      What is homologous rekombination?

5.      What are Embryonic Stem cells ?

6.      What is a blastocyst Injcetion?

7.      What is a genome?

8.      What is nuclear transfer?

9.      What is a conditional knockout

10.  What is a knockin

11. How are knockout mice made?

 

A knockout mouse is a genetically modified mouse which has one or several genes made inoperable by the insertion of a mutated gene or a disrupted form of the gene.

The generation of knockout mice is important for the analysis of the function of many unknown genes. However, complications arise when knockouts of genes result which are lethal to the animal. Embryonic lethals or death of mice at early embryonic stages is a common effect of knocking out genes which are important in development. Recently, progress has been made in creating knockout mice which are not embryonic lethal. Embryonic lethality has been minimized by the introduction of inducible knockouts which knockout the gene later in the life of the mouse (i.e. the adult stage).

(A transgenic mouse is a mouse which has foreign inserted DNA sequences, however these may not lead to a gene knockout.)

By using "models" such as animals or cells, researchers try to predict the answers they would obtain directly if they could perform the experiments in humans. Genetically manipulated animals, like humanized mouse, have proved hugely promising tools to decipher physiological processes. Since the physiology of the animal used is different from human physiology, false positives (very promising results obtained in biological models turned out negative in humans) and false negatives (programs stopped owing to poor results obtained, e.g., in the mouse, even though in humans the results would have been excellent) dramatically reduce the final quality of the research. Therefore, increasing the predictability (ability of a model to predict human physiology) through genetic manipulation is recognized as a key success factor and as the ultimate way to shorten drug development time. Obtained with classical transgenesis or homologous recombination, humanized mouse is a powerful research tool to physiology in vivo.

Pro-nuclear DNA Injection is the oldest and most common method for generation of transgenic mouse. A solution of the transgene is injected into the oocyte with a microneedle under a microscope. The micro-injected oocytes are then transplanted in a pseudopregnant mother. At birth, the newborn are then tested for the integration of the transgene. The positive animals (founder) are then intercrossed to obtain the transgenic mouse line.

When a DNA fragment is introduced in the nucleus of a cell it can integrate randomly into the genome. If the introduced DNA is identical (homologous) to a DNA sequence (locus A) present in the genome, then the integration can occur in the locus A. Based on that property it is then possible to target a mutation in a specific location of the genome. This technology is called homologous recombination.

 

A foreign sequence (e.g., a Neo cassette) can be introduced by homologous recombination in a mouse gene A. The insertion of the foreign sequence is designed to prevent the transcription of the mouse gene A. The mouse gene A is no longer expressed; the gene A is "knocked-out". If the experiment is performed with mouse Embryonic Stem cells (ES cell), injection of the recombinant Embryonic Stem cells (ES cell) into blastocysts give rise to chimeras, heterozygous and homozygous animals, also called knock out mouse.

 

A transgene B (e.g., a sequence coding for a fluorescent protein) can be introduced by homologous recombination in a mouse gene A in such a way that the transgene B transcription is driven by the promoter of the mouse gene A. The transgene B is expressed with the same pattern of expression as the mouse gene A; this strategy is called "knock-in". If the transgene B is the human counterpart of the mouse gene A, then this strategy is called humanization and the mouse a humanized mouse.

Mouse Embryonic Stem cells (ES cells) are used to establish mutant mice (knock out and knock-in mouse). Embryonic Stem cells are continuously growing cells derived from the inner cell mass of the embryo at the blastocyst stage (3.5 days post coito). These cells possess three unique features:

• They are naturally immortal. Embryonic Stem cells can grow indefinitely in vitro without the use of immortalizing or transforming agents.
  
• They are pluripotent. Embryonic Stem cells maintain the capacity for differentiation into a wide range of tissues in vitro and in vivo.
  
• They are highly tumoral. When injected in syngenic mice, Embryonic Stem cells develop teratocarcinoma tumors that comprise cells of different embryonic origins.

 

Blastocyst injection : Embryonic Stem cells reintroduced into host blastocysts can contribute to all adult tissues, including germ cells. After blastocyst injection, embryos are reimplanted in a pseudopregnant mother. The mouse obtained from injected blastocysts is made of cells of two origins (host blastocyst derived cells and injected Embryonic Stem cells) and is called a chimera Therefore, a genetic modification introduced in Embryonic Stem cells by homologous recombination can be introduced in the germ line of a chimera (mouse) and be transmitted to progeny.

 

The genome represents the entire genetic information stored in the chromosomes of an organism. It enables the fertilized egg (i.e. the zygote) or an embryonic stem cell to complete development into an adult organism containing all organs, physiological functions and structures necessary for living and reproduction. This genetic information is maintained in the nucleotide sequence of DNA, where DNA is an integrated part of the chromosomes. Molecular DNA duplication-processes guarantee the genome to be present in nearly all cells of a respective organism. The genome also serves as a sanctuary for long term storage of the genetic information established over millions of years of evolution. To this end, replica copies of each DNA strand are produced by duplication, which, if damaged through mutation, serve as blueprints for each other to correct occurring errors.

Scientists can take the nucleus out of a fertilised egg cell, and replace this with a nucleus from the patient. This reprogrammed egg cell can be used to clone more cells with the adult nucleus. The resultant cell line could then be used to treat the patient. Obviously not everyone feels comfortable about this technology, because it involves using embryonic tissue.

While knockout mice technology represents a valuable research tool, some important limitations exist. About 15 percent of gene knockouts are developmentally lethal, which means that the genetically altered embryos cannot grow into adult mice. The lack of adult mice limits studies to embryonic development and often makes it more difficult to determine a gene's function in relation to human health. In some instances, the gene may serve a different function in adults than in developing embryos.

To overcome these drawbacks, the conditional knockout approach allows researchers to delete the gene of interest in a time- and space-dependent manner. A Cre-loxP or Flip-FRT system is used to excise a critical part of the gene. Often a Cre or Flip transgenic mouse is crossed with the knockout-ready mouse with LoxP or Flip sequences flanking the critical part of the gene. Timing and space-dependence is achieved by the choice of promoter used to drive the Cre gene.

In contrast to knockout in which a gene or part of a gene is deleted, knockin is the replacement of a gene by mutant version of the same gene using homologous recombination. Knockin is very useful when establishing a disease model of a specific disease-related mutation in human gene.

Researchers begin by harvesting embryonic stem (ES) cells from early-stage mouse embryos four days after fertilization. ES cells are used because they are able to differentiate into nearly any type of adult cell, which means that if a gene is knocked out in an ES cell, the effects can be observed in any tissue in an adult mouse. In addition, ES cells grown in the lab can be used to make knockout mice as long as 10 years after they were harvested.

Gene targeting or homologous recombination is the method researchers use to specifically manipulate a gene in the nucleus of an ES cell. Typically, this is done by introducing an artificial piece of DNA that shares identical, or homologous, sequence to the gene. This homologous sequence flanks the existing gene's DNA sequence both upstream and downstream of the gene's location on the chromosome. The cell's own nuclear machinery automatically recognizes the identical stretches of sequence and swaps out the existing gene or portion of a gene with the artificial piece of DNA. Because the artificial DNA is inactive, the swap eliminates, or "knocks out," the function of the existing gene. This kind the knockout is often referred as constitutive knockout or traditional knockout, as oppose to the conditional knockout described below.

For gene trapping, the vehicle used to ferry the artificial DNA into ES cells often consists of a linear fragment of bacterial DNA. After the artificial DNA is inserted, the genetically altered ES cells are grown in a lab dish for several days and injected into early-stage mouse embryos. The embryos are implanted into the uterus of a female mouse and allowed to develop into mouse pups.

The resulting mouse pups have some tissues in which a gene has been knocked out - those derived from the altered ES cells. However, they also have some normal tissues derived from the non-altered embryos into which the altered ES cells were injected. Consequently, they are not complete knockout mice. It is necessary to crossbreed such mice to produce lines of mice in which both copies of the gene (one on each chromosome) are knocked out in all tissues. Researchers refer to such mice as homozygous knockouts.