Transgenics Flashcards
(38 cards)
When is transgenics used?
1) To study the function of the genes
2) To produce novel products
3) As model systems for human diseases or crop plants
What is transgenics?
Organisms with transgenders
Transfer a gene into some organisms
How are transgenic animals generated by microinjection of DNA into fertilized eggs?
1) DNA is injected into the nucleus of a zygote
2) The injected zygote is implanted into a pregnant female mouse
3) Each of the offspring is examined for the presence of the injected DNA to identify transgenic mice because very few zygotes actually take in this foreign DNA
How are transgenic animals generated by embryonic stem cell technology?
1) Mate mice from a dark-coloured strain to obtain embryos at the blastocyst stage
2) Culture embryonic stem cells from the inner cell mass
3) Transfect the embryonic stem cells with marker DNA
4) Inject the transfected embryonic stem cells into a blastocyst from light coloured parents
5) Implant the injected blastocyst into a light-coloured female to obtain a chimeric off-spring
6) Mate the chimera to a light coloured mouse to obtain off-spring
7) Examine the DNA from the dark coloured offspring to determine if they contain marker DNA sequences. Mice that have these marker DNA sequences are transgenic
What are chimeric animals?
Progenies of pseudopregnant females are chimeras, because these animals have two types of cells that have contributed to the formation of adult tissues, some with the transgene and some without
How are true transgenic animals obtained?
True transgenic animals may be obtained through breeding chimeras, only if some germ line cells in the chimeras have the transgene
Explain the chimeric mouse
A chimeric mouse is a mosaic: cells developed from the original mouse, but also the engineered stem cells
cells from both randomly integrate, and you hope that the engineered stem cells integrate into the germ-line.
There are cells from both the white, and black (engineered stem cells) mouse
Only dark coloured mice are possibly transgenic
Light coloured mice are not transgenic
Why are only dark coloured mice possibly transgenic?
White mouse genotype: (bl-/bl-, trans-/ trans-)
Chimeric genotype: (bl+/bl+, trans+/trans-) if genetically engineered embryonic stem cells from black mice
When you cross the white mouse with the genetically engineered mouse you can get:
–> (bl+/bl-, trans+/trans-) OR (bl+/bl-, trans-, trans-): Mice are all grey here, but only some have the transgene
The chimeric genotype may have germ cells that have the genotype: (bl-/bl-, trans-/trans-), so when you cross this with the white mouse you get:
(bl-/bl-, trans-/trans-): Mice are white with no transgene
What does it mean to knock out and knock in a gene?
Knock out: targeted gene deletion, remove a gene
Knock in: targeted gene replacement, swap in and swap out a gene
Targeted: control for where the gene goes
How can a gene be disrupted by targeted homologous recombination?
There is a chromosome that contains a p and pa arm, which are homologous arms
There is a target vector which contains an arm that is homologous to the p and an arm that is homologous to the pa, but the target vector also contains a selectable marker gene.
1) The p and pa arm in the chromosome undergo homologous recombination with the target vector (p and p) and (pa and pa).
2) The resulting chromosome contains the p and pa arm, but also contains the selectable marker gene from the target vector, which is in between the p and pa
How does positive-negative selection for targeted integration occur?
There are two ways:
1) Non-specific integration
2) Homologous recombination
Explain how the non-specific integration for positive-negative selection for targeted integration occur?
There are thymidine kinases (tk1, tk2), two DNA sequences (HB1 and HB2) that are homologous to a specific chromosomal region in the recipient cell, Neo r, which confers resistance to the cytotoxic compound G-418, and the transgene.
1) This vector is incorporated into the chromosome.
2) After incorporation, cells are selected for resistance to G-418 and glanciclovir. –> Glanciclovir is cytotoxic to cells that synthesize thymidine kinases.
3) Other non-homologous integrations (or non-specific integration) can occur and produce inserts with one or the other of the tk
4) After treatment with G-418 and ganciclovir, the cells with non-specific integration, that includes at least one tk are killed
5) Cells do not carry the thymidine kinase, because any cells that carry the tk will be killed by glanciclovir.
6) Final cells do not have the tk, but have everything else
Explain how the homologous recombination for positive-negative selection for targeted integration occur?
1) The HB1 and HB2 in the vector, as well as the HB1 and HB2 in the chromosome undergo homologous recombination.
2) The tk1 and tk2 do not undergo homologous recombination with the chromosome, and as such are not integrated at the targeted site.
3) Cells survive double selection because tk is not present, so cells will not be killed by glanciclovir
How does genetic modification with the Cre-loxP recombination system work?
Cre: enzyme
loxP: DNA
The Cre-loxP recombination system is derived from genetic elements of the bacteriophage P1
A loxP site consists of two 13 bp inverted repeats that are separated from each other by an 8 bp spacer sequence
Two remote loxP sites come together, each of which is cleaved by the Are recombinase within the spacer regions between the repeat sequences; the two DNAs exchange and join to form recombined DNA molecules
Can a loxP site have repeats in the same orientation?
The loxP site can have repeats that are inverted, separated by a spacer or have repeats that are in the same orientation, also separated by a spacer
What happens if the two loxP sites have an opposite direction?
The Cre recombinase inverts the sequence in between the two opposite loxP sites. For example, if the sequence in between goes in the right direction, it is inverted and now goes in the left direction through looping
What happens if the two loxP sites have the same direction?
The Cre recombinase excises or removes the sequence in between the two loxP sites facing the same direction through a looping mechanism
How is the Cre-loxP site used to inactivate a gene?
Cre enzyme is only expressed in certain cells, so this is a tissue specific knockout system
1) Let’s say the loxP sites have the same direction, and exon 2 is in between these two loxP sites that have the same direction,
2) The Cre recombinase comes in, and the two loxP sites line up, and exon 2 (the sequence in between the two loxP sites) is removed/degraded.
3) The resulting product is one loxP site in between exon 1 and exon 3, with exon 2 being removed, leading to the inactivated gene
4) The degraded product is exon 2, with one loxP site
How can the Cre-loxP system be used to activate a transgene?
Normally the promoter is blocked, so the gene is turned off
There are two loxP sites that have the same direction. The promoter that is blocked, is in between these two loxP sites which have the same direction
1) The Cre recombinase comes in, which allows the two loxP sites to line up, which allows for the blocked promoter to removed
2) The final product contains the three exons, the blocked promoter is removed, but there is one loxP site before exon 1 which results in an activated gene
3) The degraded product is the blocked promoter, with one loxP site
What is the tetracycline inducible system?
The Tetracycine receptor (TET) is a bacterial sequence specific DNA binding protein.
Its binding to the promoter of the tetR gene is regulated by the presence of Tetracycline
How does the tetracycline inducible system work?
In the tetracycline inducible systems the TET DNA binding domain (DBD) is fused to a mammalian transactivation domain (TA)
The synthetic tetracycline responsive promoters contain the TET-DNA binding site from the tet R promoter (TRE) inserted upstream of basal mammalian promoter elements
The TA is the activator, which binds to the DBD which is the DNA binding domain which binds to the TRE on the DNA
What is the TET-off inducible gene expression system?
The TET-off protein is a mutant, which can not bind DNA in the presence of tetracycline
Upon the removal of tetracycline TET-off binds DNA
What is special about the TET-off inducible gene expression system?
Expression plasmids are designed to respond to TET-off-TA
The TET-off system works well in tissue cultured cells, but not so well in whole organisms because:
Side effects of continuous usage of tetracycline
Degradation of tetracycline (multi-drug resistance)
How does the TET-off inducible gene expression system work?
The system contains two plasmid: one expresses the TET-off-TA fusion protein and the other contains the protein encoding sequence under the control of the TET-responsive promoter
In the presence of tetracycline: the TET-DBD and TA cannot bind to TRE, and no gene activation
In the absence of tetracycline: the TET-DBD and TA bind to the TRE, which causes gene activation
