Recombinant DNA and genetic engineering (Dr. Boucrot)

Question 1

What is genetic engineering ?

Answer 1

Genetic engineering is the direct manipulation of the genome of an organism using biotechnology.

Question 2

What can we make with genetic engineering ?

Answer 2

Artificial drugs (insulin), materials (silk), tissues (cartilages), but also the possibility to correct genetic diseases.

Question 3

What is recombinant DNA ?

Answer 3

Recombinant DNA is combing two pieces of DNA that would not normally be found together, using artificial means, rather then by genetic recombination

Question 4

Where do we place the DNA to insert it in an organism ?

Answer 4

In a vector, a vehicle used to transfer DNA to the cell. (e.g. bacterial plasmid, virus, etc.).

Question 5

How do we insert the DNA into a vector ?

Answer 5

By adapting naturally occurring enzyme (e.g. restriction endonucleases, DNA ligases, DNA polymerases, reverse transcriptases) to manipulate DNA in vitro and insert it in vivo.

Question 6

What is the advantage of working with bacteria (E.Coli K and B strains (atenuated)) ?

Answer 6

E.Coli bacteria replicate very rapidly (very 20min) and grow exponentially : 1 bacterium can grow into 5*10E21 in one day.

Question 7

Where do restriction enzymes come from ?

Answer 7

From a bacterial self defense mechanism: the bacteria recognizes short sequences of double-stranded DNA as sites for cleavage (does not cleave its own DNA because it methylates it at certain sites with methyl’s).

Question 8

What sites does E.Coli recognise for cleavage ?

Answer 8

The EcoRI site: 5’-G/AATTC-3’.

Question 9

What are isoschozomers ?

Answer 9

Enzymes that recognize the same site but cleave differently.

Question 10

What restriction enzyme can cleave a blunt end (cut straight in the middle ?

Answer 10

SmaI.

Question 11

What is a sticky end ?

Answer 11

The end of a DNA double helix at which a few unpaired nucleotides of one strand extend beyond the other.

Question 12

What is a plasmid ?

Answer 12

A plasmid is a piece of circular DNA found in bacteria that replicates autonomously and independently of the bacterial DNA (contains an origin of replication). It often contains genes (1 or 2) for resistance to antibiotics of production of toxins. It can be extracted and modified for use by centrifugation.

Question 13

How can we select the bacteria that have taken up the gene of interest ?

Answer 13

Insert a gene that will be expressed and result in production of antibodies (e.g. resistance to ampicillin).

Question 14

What are ligases ?

Answer 14

Ligases are enzymes uses to ligate, to stick the two ends of DNA together (or other substances).

Question 15

What are the steps of DNA cloning ?

Answer 15

We enzymatically insert the DNA fragment into the plasmid vector, we mix E.Coli with plasmids in presence of CaCl2, heat pulse, culture on nutrient agar plates containing ampicillin. The transmorfed cells survive and the others die. We then let the plasmid replicate and E.Coli bacteria divide.

Question 16

What is electrophoresis ?

Answer 16

Electrophoresis is a technique used to analyze DNA by placing a mixture of DNA restriction fragment in the well of an agarose or polyacrylamide gel. These gel have small pores, allowing the DNA molecules to move through them when an electric field is applied. Molecules move at a rate inversely proportional to their length. We can then subject the gel to autoradiography of incubate it with a fluorescent dye observe signals corresponding to DNA bands.

Question 17

What is southern blotting ?

Answer 17

After the DNA fragments have migrated far enough, they are treated with restriction enzymes and either heated or soaked in an alkali solution (NaOH) do separate the molecule into two strands (only ssDNA can be transferred). The gel used for electrophoresis is then placed atop a thin sponge wick resting in a dish of salt solution, and a special filter (typically nitrocellulose) is placed on top of the gel. A stack of absorbent material (typically paper towels) is placed on top of this stack. The absorbent material draws the salt solution from the dish into the wick and through the gel by capillary action, which transfers the DNA fragments into the filter. The procedure is called a “Southern blotting” after the scientist Eric Southern who invented the procedure. The filter now contains the DNA fragments in the same pattern as the gel, but is more easily manipulated.
The filter is placed in a standard “seal-a-meal” bag, containing a solution of radioactively-labelled DNA or RNA probe (complementary sequence) for a particular gene. The probe thus binds to the filter only where a complementary DNA sequence is located. After washing to remove unbound probe, a piece of X-ray film is placed over the hybridized filter and exposed for several hours to several days. The radioactive label produces a black band on the film where it has stuck to the complementary DNA, producing an autoradiogram. If a labelled size marker has been used, the exact sizes of the fragments can be determined.

Question 18

What criteria are important in choosing a vector ?

Answer 18

It’s size and replication capacity and rate. Only about 20kb can be cloned with plasmids, yet these grow exponentially, whereas the YAC (Yeast Artificial Chromosome) can accommodate as much as 1000kb but does not divide as quickly.

Question 19

What is transient transfection ?

Answer 19

In transient transfection, the vector (usually a plasmid) is inserted into the nucleus of the cell, but does not get integrated into the chromosome. The plasmid has a reporter gene that allows the scientist to monitor the expression, usually within 1-2 days post-transfection. Therefore, for a short period of time, there are many copies of the foreign gene in the nucleus, and the cDNA will temporarily be expressed. However, as the gene is not incorporated into the genome, the transfected gene does not pass to future generations of the cell. Highly supercoiled DNA appears to be superior for transient transfections.

Question 20

What is stable transfection ?

Answer 20

In stable transfection, the vector (usually a plasmid) DNA is integrated into the chromosomes, or as an episome a separate piece of nuclear DNA, and gets passed on to future generations of the cell. This is a much rarer occurrence and complex to perform, as sometimes only a portion of the plasmid gets integrated, which may not contain the gene of interest. All stable transfections start out as a transient transfection, with selectable markers that are able to distinguish any cells that have successfully integrated the gene into their genome. A common method used is to co-transfect the gene of interest with another gene for antibiotic resistance, and treat the transfected cells with the antibiotic. Repeating antibiotic treatment for long-term cell cultures results in the expansion of stably-transfected cells. Linear DNA appears to be better for stable transfection, although its uptake is lower than supercoiled DNA.

Question 21

What are GMOs ?

Answer 21

GMOs are organisms that have their genetic structure changed artificially (bacteria, plants, fish, animals, humans).

Question 22

What is the total surface area of all the “GMO farms” in the USA ?

Answer 22

181 million hectares.

Question 23

What is a KO (Knock Out) mouse ?

Answer 23

A KO mouse is a laboratory mouse in which researchers have inactivated, or “knocked out,” an existing gene by replacing it or disrupting it with an artificial piece of DNA.

Question 24

What is Cre-recombinase ?

Answer 24

Cre-recombinase is a tyrosine recombinase enzyme derived from the P1 Bacteriophage. The enzyme uses a topoisomerase I like mechanism to carry out site specific recombination events.

Question 25

What are LoxP sites ?

Answer 25

LoxP (locus of X(cross)-over in P1) sites are 34-base-pair long recognition sequences consisting of two 13-bp long palindromic repeats separated by an 8-bp long asymmetric core spacer sequence. The asymmetry in the core sequence gives the loxP site directionality, and the canonical loxP sequence is ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT. The loxP sequence does not occur naturally in any known genome other than P1 phage, and is long enough that there is virtually no chance of it occurring randomly. Therefore, inserting loxP sites at deliberate locations in a DNA sequence allows for very specific manipulations

Question 26

How does Cre-Lox recombination work ?

Answer 26

Cre-Lox is a system that can be used to introduce gene deletions, inversions, and translocations on specific target sites. In many cases, systematically deleting the gene would produce an embryonic lethal phenotype, rendering an experiment useless because we couldn’t observe the effect of the deletion. The Cre-Lox system allows us to have temporal and tissue-specific control on where to have the deletions.

Question 27

What is CRISPR /Cas9 ?

CRISPR = Clustered Regularly Interspaced Short Palindromic Repeat

Answer 27

CRISPR was identified in a bacterial/archean defense mechanism against plasmids and phages. CRISPR are sections of genetic code containing short repetitions of base sequences followed by spacer DNA segments.
CRISPR/Cas9 is an RNA-guided gene editing platform derived from streptococcus pyogenes using an endonuclease (Cas9) and synthetic guide RNA to introduce a double strand break at a specific location within the genome.

Question 28

What is somatic gene therapy ?

Answer 28

Somatic cell gene therapy changes, fixes or replaces genes in just one person. The targeted cells (somatic cells = all but sperm and eggs) are the only ones affected, the changes are not passed on to that person’s offspring