1. Gene Knockout
査向东
安徽大学生命科学学院

2. Definition of targeted gene knockout : The introduction of a null mutation in a gene by a designed alteration in a cloned DNA sequence that is then introduced into the genome by homologous recombination and replacement of the normal allele.
The technology's origins lie in a natural phenomenon called homologous recombination.

3. The Nobel Assembly at Karolinska Institutet has awarded The Nobel Prize in Physiology or Medicine for 2007 jointly to Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their discoveries of "principles for introducing specific gene modifications in mice by the use of embryonic stem cells."
Mario Capecchi from the University of Utah in Salt Lake City, Martin Evans of Cardiff University in Wales and Oliver Smithies of the University of North Carolina at Chapel Hill, share the €1.1-million (US$1.5-million) award. As is now often the case with this Nobel prize.

4. Gene Targeting Pioneers Win Nobel Prize For Discoveries In Embryonic Stem Cells And DNA Recombination
ScienceDaily (Oct. 8, 2007)
Modification of genes by homologous recombination
Embryonic stem cells – vehicles to the mouse germ line
Two ideas come together – homologous recombination in ES cells
Birth of the knockout mouse – the beginning of a new era in genetics
Gene targeting is used to study health and disease, or to uncover functions of genes.

5. Method
Knockout is accomplished through a combination of techniques, beginning in the test tube with a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell culture. Individual cells are genetically transformed with the DNA construct. Often the goal is to create a transgenic animal that has the altered gene. If so, embryonic stem cells are genetically transformed and inserted into early embryos. Resulting animals with the genetic change in their germline cells can then often pass the gene knockout to future generations.
The construct is engineered to recombine with the target gene, which is accomplished by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene in most cases will be translated into a nonfunctional protein, if it is translated at all.

6. Because the desired type of DNA recombination is a rare event in the case of most cells and most constructs, the foreign sequence chosen for insertion usually includes a reporter. This enables easy selection of cells or individuals in which knockout was successful. Sometimes the DNA construct inserts into a chromosome without the desired homologous recombination with the target gene. To avoid isolation of such cells, the DNA construct often contains a second region of DNA that allows such cells to be identified and discarded.

7. In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection are performed until every targeted gene is knocked out. Selective breeding may be required to produce homozygous knockout animals.
A conditional knockout allows gene deletion in a tissue or time specific manner.
Knock-in is similar to knock-out, but instead it replaces a gene with another instead of deleting it.

8. Briefly
Engineering of a targeting vector and its subjection to homologous recombination in embryonic stem (ES) cells
injection of the targeted ES cells into mouse blastocyts for further germ line transmission.

9. Figure 1: Gene targeting for knockout mice

10. Knock-out mice
Gene targeting has already produced more than five hundred different mouse models of human disorders, including cardiovascular and neuro-degenerative diseases, diabetes and cancer. (Credit: iStockphoto/Andrei Tchernov ）

11. Knockout Mouse Copyright 2002 Department of Biology, Davidson College, Davidson, NC 28036
Step 1. Isolate developing embryo at blastocyst stage. This embryo is from a strain of mice with gray fur.

12. Step 2. Remove embryonic stem cells from gray-fur blastocyst
Step 2. Remove embryonic stem cells from gray-fur blastocyst. Grow stem cells in tissue culture

13. Step 3. Transfect stem cells with homologous recombination construct
Step 3. Transfect stem cells with homologous recombination construct. Select for homologous recombination by growing stem cells in neomycin and gancyclvir.
+ neomycin
+ gancyclovir

14. Step 4. Remove homologously recombined stem cells from petri dish and inject into a new blastocyst that would have only white fur.

15. Step 5. Implant several chimeric blastocysts into pseudo-pregnant, white fur mouse.

16. Step 6. Mother will give birth to a range of mice
Step 6. Mother will give birth to a range of mice. Some will be normal white fur mice but others will be chimeric mice. Chimeric mice have many of their cells from the original white fur blastocyst but some of their cells will be derived from recombinant stem cells. Fur cells from recombinant stem cells produce gray patches which are easily detected.

17. Step 7. Mate the chimeric mice with wild-type white fur mice
Step 7. Mate the chimeric mice with wild-type white fur mice. If the gonads of the chimeric mice were derived from recombinant stem cells, all the offspring will have gray fur. Every cell in gray mice are heterozygous for the homologous recombination.

18. Step 8. Mate heterozygous gray mice (+/ H) and genotpye the gray offspring. Identify homozygous recombinants (H / H) and breed them to produce a strain of mice with both alleles knocked out. The pure breeding mouse strain is a "knockout mouse".

19. Figure 4. 17 Conditioned gene knock-out
Figure 4.17   Conditioned gene knock-out. A normal gene is replaced by a copy that is flanked by loxP sites, which are repeated sequences. A separate gene, cre recombinase, is inserted elsewhere adjacent to a specific promoter. When the cre recombinase is activated, recombination between the loxP sites results in removal of the gene. The promoter might be inducible by administration of a hormone or chemical, in which case timing of the knock-out is under control. Alternatively, it might be a tissue-specific promoter, in which case knock-out will only occur in specific tissues.

20. Cre recombinase, often abbreviated to Cre, is a Type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. The enzyme does not require any energy cofactors and Cre-mediated recombination quickly reaches equilibrium between substrate and reaction products. The loxP recognition element is a 34 base pair (bp) sequence comprised of two 13 bp inverted repeats flanking an 8 bp spacer region which confers directionality. Recombination products are dependent on the location and relative orientation of the loxP sites. Two DNA species containing single loxP sites will be fused whilst DNA between loxP sites in the same orientation will be excised in circular form and DNA between opposing loxP sites will be inverted with respect to the rest of the DNA.
Cre recombinase is used as a tool to modify genes and chromosomes. In this approach the Cre recombinase is used to delete a segment of DNA flanked by LoxP sites (aka 'floxed') in an experimental animal. It has been used to generate animals with mutations limited to certain cell types (tissue-specific knockout) or animals with mutations that can be activated by drug administration (inducible knockout) in a number of transgenic species.[1] The availability of transgenic lines with tissue specific or inducible Cre expression permits researchers to inactivate or activate a gene of interest simply by breeding a floxed animal to pre-existing Cre-transgenics.
From Wikipedia, the free encyclopedia

21. Exercises
Define and explain
Holiday junction, gene conversion,
gene knockout
Distinguish Genetic recombination and recombinant DNA technology.What are the benefits of genetic recombination?