Researchers have identified a gene in humans that (when mutant) causes severe dw

Question

Researchers have identified a gene in humans that (when mutant) causes severe dwarfism and mental retardation. This disorder is inherited in an autosomal recessive manner, and the mutant allele is known to be a loss-of-function mutation. The same gene has been found on mice, although a mutant version of the gene has not been discovered in mice. To develop drugs and an effective therapy to treat this disorder, it would be extremely useful to have a mouse model of the disorder. In other words, it would be desirable to develop a strain of mice homozygous for a loss-of-function mutation in the mouse gene. Experimentally, how would you develop such a strain? List all of the steps necessary to accomplish this

Explanation / Answer

* The normal gene from the mouse is closed. To clone the normal gene, forward and reverse primers that flank the gene are designed, and then, using PCR method, primer extension is done. This gives the normal mouse gene

* Through site directed mutagenesis, multiple cloning site MCS may be inserted within this normal gene. Noemycin resistance gene (NeoR) is inserted into the normal gene to inactivate it

* Thymidine kinase gene should be cloned next to it. It acts as a lethal gene. Cells expressing the gene can be killed using ganciclovir, an antiviral drug

* The DNA segment should now be introduced into the mouse embryonic stem cells, and grown in the presence of neomycin and gancyclovir. This selects homologous recombinants, all other cells may either do not grow or are killed

* The surviving stem cells are injected into early embryos, which develop later into chimeras

* The chimeric mice are identified by their morphological features that differ from the normal mice

* Heterozygous individuals that have one copy of normal gene and the other with NeoR insert are identified, and bred to each other. This gives rise to offspring that are homozygous for the NeoR insert genes

* The homozygotes are knok-out animals since the gene function is inactivated by NeoR gene insertion. Therefore, such mice would exhibit dwarfism and mental retardation

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