FLOW CHART FOR THE PRONUCLEAR INJECTION TECHNIQUE

The pronuclear injection technique is suitable for adding genes to the genome in situations where the endogenous genetic makeup is not important, for example when dominant effects or promoter/enhancer activities are studied. For overexpression, a promoter/enhancer that directs expression to a particular organ or timepoint during development is coupled to the gene or cDNA of interest, and for gene regulation studies, the promoter/enhancer of interest is coupled to a reporter gene.

A purified DNA construct is injected into fertilized mouse eggs at the one-cell stage.

DNA integration in the genome is random, and the number of integrated copies ranges

from one to more than a hundred. In most cases, multiple copies of the introduced DNA are integrated in tandem at a single chromosomal site.

After injection, eggs are transferred into oviducts of pseudopregnant females, where they develop further, and offspring is born approximately 20 days later. Mice that have the transgene integrated in their genome are identified by DNA analysis. For a more detailed account of the pronuclear injection technique, please see Hogan et al., 1994.

Below follows a schematic representation of a pronuclear injection project carried out by KCTT:

FLOW CHART FOR THE ES CELL TECHNIQUE

The key features of the ES (embryonic stem) cell technique can briefly be summarized as follows.

Specific mutations, for example inactivation or modification of a gene, can be introduced into the genome by homologous recombination, which leads to the replacement of the endogenous gene fragment by the introduced, engineered fragment.

Because homologous recombination is a rare event, special selection and screening strategies are needed. Usually a specific selectable marker gene is included in the targeting DNA construct.

A targeting construct, which is made from mouse 129 strain DNA, is introduced into totipotent mouse embryonic stem (ES) cells (isolated from 129 mice). Cells that have integrated the introduced DNA in their genome are selected using a drug that kills cells which have not taken up the marker gene. Selected clones are then analyzed by restriction enzyme digestion and Southern blot or by PCR analysis to identify the clones where homologous recombination has occurred. Positive clones are then expanded.

ES cells from correctly targeted clones are introduced into mouse embryos at the blastocyst stage by injection, and injected blastocysts are transferred into the uteri of pseudopregnant females. Chimeric (mosaic) mice consisting of cells originating from the blastocyst and from ES cells are born approximately 17 days after transfer. Chimeric mice are then tested for their ability to transmit the introduced mutation to the next generation (germline transmission).

For a more detailed account of this technology you can look up the following literature:

Below follows a schematic representation of an ES cell project carried out by KCTT: