Cloning via Somatic Cell Nuclear Transfer
Do Now: Name the three things in the picture below.
Answers: You can see the:
Nucleus that's been aspirated out of the egg cell.
Egg cell from a donor individual. The egg cell above is enucleated.
Zona pellucida - a protein coat around the egg.
The larger of the two pipettes, with a smooth, rounded tip so as to not puncture the egg.
Uses suction to hold the egg in place, so the nucleus can be removed and another injected.
Piezoelectric drilling pipette
The smaller of the two pipettes, with a blunt tip.
It is piezoelectric so it can drill a hole through the zona pellucida.
1) Roslin Method
Enucleated egg + whole somatic cell
Electrical pulse used to fuse the membranes of both cells together + stimulates development.
2) Honolulu Technique of SCNT
Enucleated egg + somatic cell nucleus (NOT whole cell)
Electrical pulse used to activate cell to undergo mitosis. Divides to become early embryo (called 'blastocyst').
Genetic Modification via Transgenesis
Step 1: Identification
Compare the elephant's known gene of interest against the mammoth DNA sequence to find the mammoth's gene of interest.
Search up the woolly mammoth's complete genome sequence in DNA databases such as GenBank by NCBI and ENA (European Nuclear Archive).
Links to Mammoth's Nuclear DNA Sequence
Link to the article above: https://www.cell.com/current-biology/fulltext/S0960-9822(15)00420-0#secsectitle0035
To actually download the complete sequence of the woolly mammoth, click on the website below and type the following accession number "PRJEB7929"
Then search up the elephant's known gene of interest (e.g. elephant haemoglobin gene) in gene databases like Gene by NCBI. Compare the known elephant sequence against the mammoth's entire genome to find the mammoth's haemoglobin gene.
Step 2: Isolation
Now that you've identified the mammoth's gene of interest (e.g. haemoglobin), you know the sequence you need to isolate. You should use the technique called PCR (polymerase chain reaction), to isolate this specific gene sequence.
Step 1: Denaturation (95°C) - to separate DNA into single strands.
Step 2: Annealing (55°C) - specific primers you designed to target the gene sequence will bind to the correct complementary sequence on your single stranded DNA.
Step 3: Extension (72°C) - Taq polymerase extends the DNA primers by adding bases using the complementary base pairing rule.
Steps 1-3 are repeated many times, to amplify this specific gene sequence.
Now that you have amplified your mammoth gene sequence from the rest of the mammoth DNA, you've got to purify it to cleanse it from the other substances involved in the PCR reactions. These other substances include other DNA sequences, primers, enzymes, and other reagents. You will purify your gene sequence using gel electrophoresis.
This technique separates DNA sequences according to size. Size is measured using a 'DNA ladder; as a reference. When you've separated your gene of interest from the rest of the DNA
Gel electrophoresis https://www.addgene.org/protocols/gel-purification/
Sanger Sequencing / DNA Sequencing
Four reaction tubes are created, all with a small sample of your purified DNA.
ddNTPs for adenine is added to tube 1
ddNTPs for thymine is added to tube 2
ddNTPs for cytosone is added to tube 3
ddNTPs for guanine is added to tube 4
You run each tube through the steps of PCR, but instead this time, ddNTPs are randomly added throughout the sequence. For example, in tube 1 instead of DNA polymerase pairing dNTP of adenine to a thymine, it randomly adds a ddNTP of adenine to the thymine.
Once the ddNTP is added, the sequence is terminated, and DNA polymerase can no longer extend that strand of DNA. This results in a sample of DNA fragments of different lengths.
Samples from all 4 tubes are run on a gel, and DNA fragments are separated by length via gel electrophoresis. The sequence is read from in the direction of the shortest fragment to the longest fragment.
OR another way is to run the negatively charged DNA in a capillary tube. They separate according to size, and are read one by one as the ddNTP fluoresces a unique colour.
Step 3: Transfection
Transfection is the introduction of foreign DNA into mammalian cells. One way this can be done us by using the CRISPR-Cas9 bacterial system as endogenous scissors to cut out the elephant gene you are replacing.
Homology directed repair within the elephant's cell can insert your gene of interest into the site cut by CRISPR-Cas9. Click this link to watch the awesome video below: http://www.crisprtx.com/gene-editing/crispr-cas9