Chapter 20: Molecular Technologies

Recombinant DNA Technology can be used to:

• Investigate relationship between DNA sequence and phenotype

• Study gene structure and gene expression

• Gene therapy, screening for human diseases, recombinant vaccines

• Producing transgenic plants and animals in agriculture and research

What is Gene Cloning and what is it used for?

Gene cloning: the process of making many copies of a gene

• Gene cloning helps researchers understand contribution of gene to cell structure and function

What are the two types of DNA involved in gene cloning?

• Chromosomal DNA

  • Source of the gene or DNA segment of interest from the species that carries the gene

• Vector DNA

  • Serves as the carrier of the DNA segment to be cloned

  • Vectors originally derived from plasmids and viruses

What is the role of Plasmids in Gene Cloning?

• Most vectors are plasmids – originally from bacteria

• Small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA

• Unique sites where pieces of target DNA can be inserted

• They also carry an antibiotic resistance gene, allowing bacteria to survive exposure to antibiotics

What is the role of Restriction Enzymes in Gene Cloning?

• Key step in cloning is insertion of chromosomal DNA into a plasmid or viral vector

• Can be accomplished by cutting and pasting DNA fragments

• The enzymes used to cut or digest DNA are known as restriction endonucleases or restriction enzymes

• These bind to specific DNA sequences and then cleave the DNA at two defined locations, one on each strand

• Originally made by bacterial cells to protect them from invasion by foreign DNA

Definition of Gene Cloning

Refers to molecular methods that produce many copies of a gene

Definition of Reproductive Cloning

Refers to methods that produce two or more genetically identical individuals

What was the first mammalian cloning and how was it done?

In 1997, Ian Wilmut and colleagues at the Roslin Institute announced the first mammalian cloning

• A sheep, “Dolly”

Strategy for cloning Dolly:

1. Mammary gland cells were collected and grown in the lab

2. Using electrical pulses, a mammary cell was fused with a nucleus-free egg from a second sheep

3. The zygote began to develop

4. The embryo was implanted into a surrogate mother sheep

5. 148 days later, Dolly was born

Why was Dolly possibly “genetically older” than she actually was?

• Dolly’s DNA were “older”

• At 3 years, the length of her telomeres were similar to a 9 to 10 year old sheep

• Developed a progressive lung disease called sheep pulmonary adenomatosis

• Died when she was 6 years old

Definition of Stem Cells

Supply the cells that construct our bodies from a fertilized egg

• In adults, stem cells also replenish damaged cells

What are the two common characteristics of Stem cells?

1. Ability to divide

2. Can differentiate into one or more specialized cell types

What are the four types of stem cells?

• Totipotent stem cells can give rise to all cell types

• Pluripotent cells (such as embryonic stem cells or ES cells) can differentiate into almost every cell, but can’t give rise to an entire, intact individual)

• Multipotent cells can differentiate into several cell types

• Unipotent cells can only differentiate into one cell type

Definition of Gene Editing

• Experimentally altering the sequence of a gene is called gene mutagenesis or gene editing

What is the CRISPR-Cas system in bacteria?

The CRISPR-Cas gene system provides bacteria with a defense against invasion by bacteriophages

• Crispr gene has a group of clustered, regularly interspaced, short, palindromic repeats; hence the name CRISPR

• The repeats are interspersed with short, unique sequences, which are called spacers

How was CRISPR-Cas modified to be used in gene editing technology?

• Researchers created a modified version of tracrRNA called the guide RNA (gRNA)

• Instead of viral DNA, this gRNA has a sequence complementary to the target sequence in a gene to be mutated

• This gRNA then binds to the Cas9 protein, similar to that in bacteria

• Inject this complex into an individual, which is then guided it to the gene of interest

• The complex makes necessary break in this target gene