6 - Gene Manipulation I

Question 1

What three types of DNA are found after ooplasmic transfer?

Answer 1

Parent, mDNA, donor mDNA

Question 2

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

Answer 2

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

Question 3

What is a transgene?

Answer 3

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.

Question 4

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

Answer 4

True

Question 5

What is the enviropig?

Answer 5

A pig engineered to with transgenes to produce less phosphorous pollution

Question 6

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

Answer 6

Calcium precipitation.

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

Question 7

What is electroporation?

Answer 7

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)

Question 8

What is lipofection?

Answer 8

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.

Question 9

What is microinjection?

Answer 9

Method of DNA insertion

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

Question 10

What method of DNA insertion has the most success?

Answer 10

Viral vectors

Not always wanted for the tendency to introduce mutations

Question 11

What are gene knock-downs?

Answer 11

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

Question 12

What must be made for a homologous recombination insert?

Answer 12

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

Question 13

What is a negative selection marker?

Answer 13

Some distance from DNA construct insert, contains reporter gene?

Question 14

What is a positive selection marker?

Answer 14

Reporter gene within DNA construct insert

Question 15

What is nuclear transfer and what are the steps?

Answer 15

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

Question 16

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

Answer 16

A consequence of telomere shortening

Question 17

What is human germline genetic modification?

Answer 17

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)

Question 18

What are three methods of obtaining transgenic animals?

Answer 18

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

Question 19

What are five uses for transgenic animals?

Answer 19

• 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

Question 20

What are 5 methods of inserting DNA into a cell?

Answer 20

• Calcium precipitation
• Electroporation
• Lipofection
• Microinjection
• Viral vectors

Question 21

What is calcium precipitation?

Answer 21

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.

Question 22

What is electroporation?

Answer 22

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

Question 23

What is lipofection?

Answer 23

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

Question 24

What is microinjection?

Answer 24

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.

Question 25

What are viral vectors?

Answer 25

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

Question 26

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

Answer 26

A promoter and enhancer

Question 27

What is a knock-out?

Answer 27

When a gene is deleted from the genome.

Question 28

What are knock-ins?

Answer 28

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

Question 29

What is a knock-down?

Answer 29

Using RNA interference to downregulate the expression of a gene

Question 30

What is homologous recombination?

Answer 30

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

Question 31

What is non-homologous end joining?

Answer 31

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)

Question 32

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

Answer 32

non-homologous recombination pathway Because this is the pathway that dominates in mice and humans

Question 33

What are the components of a homologous recombination DNA construct?

Answer 33

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

Question 34

What are the steps of homologous recombination?

Answer 34

- 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)

Question 35

What are the steps of non-homologous recombination?

Answer 35

- 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)

Question 36

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

Answer 36

- 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

Question 37

What will happen if a knockout gene is dominant?

Answer 37

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

Question 38

What will happen if a knockout gene is recessive?

Answer 38

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

Question 39

What are 4 limitations of knockouts?

Answer 39

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

Question 40

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

Answer 40

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.

Question 41

What is chemical mutagenesis?

Answer 41

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.