Biotechnology methods + therapeutic applications

What is transfection?

The process of introducing nucleic acids into cells by non-viral methods

What are the types of transfection methods?

Chemical or physical

What is transduction?

The process of introducing nucleic acids into another cell via a viral vector

What does chemical transfection involve?

Neutralisation of the negative charge of DNA by the transfection reagent, which facilitate its uptake by cells via endocytosis

What is lipofection?

transfection method, involving positively charged lipid based reagents to coat negative DNA, enabling it to enter cells by endocytosis.

What are some examples of physical transfection methods?

Electroporation, micro injection

What is electroporation?

Physical transfection, uses electrical pulse to create temporary pores into cell membrane, which nucleic acids can pass through into cells

What is microinjection?

Transfection used to introduce nucleic acids directly into either the cytoplasm or nucleus, using a glass needle

Describe viral transduction

First, virus particles containing the required exogenous DNA must be generated through transfection of the packaging cell line.

Second, the virus particles are then used to transduce the cells in which the exogenous DNA is required.

What do we use nucleic acid transfer methods for?

• gene/protein function studies

• Biomedical research

• Create transgenic animals or cells

• Recombinant protein production

What does nucleic acids refer to?

DNA and RNA

What is recombinant protein production

• biotherapeutics

• Drugs made from biological systems by telling cells what to do

RNA interference (RNAi)

To reduce (knock down) gene or protein expression

What is gene knock out?

Abolish expression of a gene

What is gene knock in?

Insert addition DNA sequence into the genome

What are uses of CRISPR - mediated gene editing?

• Commonly used for gene knock out

• Also used for gene knock in

• Adaptations of the method exist for gene silencing, DNA labelling, altered gene expression

What method is usually used for generating transgenic embryos or animals?

Microinjection

Steps of microinjection to produce transgenic cell

1. Microinjection of exogenous DNA into fertilised egg

2. Microinjection of modified ES cells

3. CRISPR gene editing of fertilised egg

What does transgenic mean?

Contains genetic material from unrelated organism

Stages of drug discovery

1. Target ID - identify biological targets and candidate drugs to hit target

2. Hit ID - find drugs or molecules that interact with target and stop it working

3. Lead generation and optimisation

Stages of drug development

1. Preclinical developments - drug testing animals

2. Clinical development - trails 1-4 with humans

3. Commercialisation - manufacturing from animal systems

What is used for biotherapeutic production from mammalian cell cultures?

Stirred tank bioreactors, which mostly suspension cells are used, but adherent cells can be used too by growing on microcarriers

What animals have been approved for biotherapeutics?

Goats, rabbits and chickens