6 - Gene Manipulation I Flashcards Preview

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Flashcards in 6 - Gene Manipulation I Deck (41):

What three types of DNA are found after ooplasmic transfer?

Parent, mDNA, donor mDNA


How can Leber congenital amaurosis (LCA) be treated with gene therapy? What can a side effect of this be?

Mutated RPE65 gene can cause LCA.

Replacing the mutated gene with a transgene by injection into the retina improved vision (from blind to sight in some cases)

Prolonged distant injection can create a 'second fovea,' distorting vision


What is a transgene?

A transgene is a gene or genetic material that has been transferred naturally, or by any of a number of genetic engineering techniques from one organism to another. The introduction of a transgene has the potential to change the phenotype of an organism.


True or false? Transgenes can be used to make human proteins in different organisms.



What is the enviropig?

A pig engineered to with transgenes to produce less phosphorous pollution


What is the oldest method of DNA insertion into the cell?

Calcium precipitation.

DNA is incorporated by endocytosis. Not always successful because cellular enzymes degrade the DNA in the endosome


What is electroporation?

DNA into cells

The cells and DNA are put under an electric current, this allows the membrane to open let DNA in (often kills the cells though)


What is lipofection?

Cheapest and most commonly used method of DNA insertion into cell.

Makes complexes of DNA with micelles, which have the ability to cross the membrane. Not a target for nucleases.


What is microinjection?

Method of DNA insertion

Directly injecting DNA or RNA into the nuclease (low success rate)


What method of DNA insertion has the most success?

Viral vectors

Not always wanted for the tendency to introduce mutations


What are gene knock-downs?

Silencing genes. No successful method of knock-downs for humans


What must be made for a homologous recombination insert?

- Create DNA construct that you want to insert (should contain a reporter gene and negative marker)


What is a negative selection marker?

Some distance from DNA construct insert, contains reporter gene?


What is a positive selection marker?

Reporter gene within DNA construct insert


What is nuclear transfer and what are the steps?

The way to create an identical copy of a cell

1. Enucleate a host oocyte
2. The nuclear donor cell si fused with the enucleated oocyte or the nucleus of the nuclear donor cell is dissected and transferred into the enucleated oocyte
3. donor nuclei are obtained from either stem cells or differentiated adult cells


Why do somatic cell-derived nuclei (eg. nuclear transfer cells) often exhibit cellular senescence (aging)?

A consequence of telomere shortening


What is human germline genetic modification?

Ooplasmic transfer from healthy donor oocytes to parent oocytes with history of implantation failure.

This results in birth of babies who carry DNA from three people (maternal, paternal and the ooplasm donor).

The mitochondrial heteroplasmy can be also obtained by pronuclear transfer (the egg nucelus and sperm nucleus are placed in the enucleated donor egg)


What are three methods of obtaining transgenic animals?

1. Germline modifications of gametes
2. Microinjection of DNA or gene constructs into zygotes (unicellular embryos)
3. Incorporating modified cells, including embryonic stem (ESC) cells into later stage embryos


What are five uses for transgenic animals?

- Model for human disease
- Study development and function of human genes
- Synthesis of proteins
- Transplantation donors
- Speed up and reduce cost of animals raised for food


What are 5 methods of inserting DNA into a cell?

- Calcium precipitation
- Electroporation
- Lipofection
- Microinjection
- Viral vectors


What is calcium precipitation?

A method of DNA insertion into a cell.

DNA is precipitated with calcium salts and precipitated material is incorporated into endosomes by endocytosis, then released into the cytoplasm and subsequently enters the nucleus. This method is fairly simple but very inefficient, mainly because much of the DNA is degraded within the endosome.


What is electroporation?

A method of inserting DNA into a cell

Cells and foreign DNA are put together in solution and electric current is applied, which opens the pores in the plasma membrane, allowing entry of the DNA


What is lipofection?

A method of inserting DNA

Formation of complexes involving cationic lipid molecules and anionic DNA. Uses micelles to pass DNA through the plasma membrane and protect against the action of nucleases


What is microinjection?

A method of inserting DNA into a cell

DNA is directly injected into the nucleus, method requires specialized equipment and skilled person. It is widely used for insertion of gene vectors into fertilized oocytes.


What are viral vectors?

Modified viruses used as a carrier of DNA into cells. This is the most efficient method of introducing foreign DNA into a cell.


What must a DNA construct include to allow it to be expressed?

A promoter and enhancer


What is a knock-out?

When a gene is deleted from the genome.


What are knock-ins?

Gene insertion into specific loci. An altered gene with point mutations is often inserted.


What is a knock-down?

Using RNA interference to downregulate the expression of a gene


What is homologous recombination?

A cellular DNA repair pathway that normally repairs double-strand DNA breaks. It also occurs during meiosis.

Nucleotides exchange between identical or similar DNA sequences


What is non-homologous end joining?

A DNA repair pathway that fuses the broken ends in a more error prone way than homologous recombination (this pathway prevails in humans and rats)


Is homologous or non-homologous recombination a better pathway for inserting a foreign gene?

non-homologous recombination pathway

Because this is the pathway that dominates in mice and humans


What are the components of a homologous recombination DNA construct?

- Reporter gene (eg. fluorescent gene)
- Negative marker outside the region of sequence between the vector and the targeted locus


What are the steps of homologous recombination?

- Make DNA construct that you want to insert into the chromosome in place of the wild-type allele
- Insert DNA construct into the cell, where it aligns with the targeted locus.
- Select cells that have undergone homologous recombination with the addition of antibiotics to the growth medium (positive selection)


What are the steps of non-homologous recombination?

- The negative selection marker will be included in the recombinant (as the recombination occurs randomly)
- The chromosome containing the thymidine kinase gene will program cell death (negative selection)


What are the steps of creating a knock-out mouse?

- Isolate a mouse embryo at blastocyst stage
- Make a cell culture from the embryonic stem cells
- Transfect colony with homologous recombination construct, remove non-transfected cells with antibiotics
- Inject homologously recombined stem cells into a white mouse blastocyst to make a chimeric embryo
- Implant chimeric blastocyst into pseudopregnant mouse (mouse that was mated with vasectomized male)
- Offspring obtained from embryos with recombinant stem cells reporter gene marker (such as gray patches)
- Cross chimeric mice with wild type to produce heterozygous for the homologous recombination) mice
- Mate heterozygous gray mice (+/H) and genotype the gray offspring. Identify homozygous recombinant (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


What will happen if a knockout gene is dominant?

Both homozygous and heterozygous offspring show the loss of function for the deleted gene. Heterozygous mice can show partial loss.


What will happen if a knockout gene is recessive?

Only homozygous offspring show the loss of function for the deleted gene


What are 4 limitations of knockouts?

- 15% are lethal
- May be different for mice and humans
- Lack of change due to compensation
- Knock outs can vary between labs


How does the procedure of doing knock-outs differ from knock-ins?

The endogenous gene is not deleted in knock-in mice but usually exchanged with one harboring a mutation of interest. It is believed that knock-ns are representing more closely physical conditions observed in nature, than those obtained by knock-out.


What is chemical mutagenesis?

The old method of doing knock-outs

Use N-ethyl-N-nitrosourea (ENU), or another mutagen, to produce random mutations in mice and rats (or drosphila). ENU is an alkylating agent that transfers its ethyl group to nitrogen or oxygen radicals in DNA resulting in base mispairing with consewuent base pair substitution.