Biotech Exam 3 Study

Type of breeding used to enhance the genetic features of domesticated animals

Selective breeding

What selective breeding developed

Pure breeding lines

Why selective breeding was not the best

Time consuming, costly, unable to introduce new genetic traits easily

Foreign DNA carrying gene for a certain trait

Transgene

Transgenes being introduced into an animal

Transgenesis

How Transgenesis is used now

Studying gene expression, establishing animal model systems for human disease, pharming

Pharming

Using mammary gland to produce pharmaceuticals in milk

Transgenic mice

Developed and perfected in mice in the early 80s

Three methods to introduce DNA into mice

Retroviral vectors infecting embryonic cells, microinjection into enlarged sperm nucleus of fertilized egg, introduce genetically engineered embryonic stem cells into an early stage embryo

What the federal animal welfare act does

Sets the standards around housing, feeding, cleanliness, and medical care of research animals

What institutional review boards do

Exist at every research institution, require researchers to prove the need to use animals, select the most appropriate species, and devise a plan for using as few animals as possible

The guide for the care and use of laboratory animals

Reduce number of higher species used, replace animals with alternative models when possible, refine tests for humane conditions

Steps to the retroviral vector method

Make defective retrovirus carrying transgene, infect 8-cell stage embryo, implant into pseudopregnant foster mother, birth of pups, test germline, establish transgenic line

Advantage of the retroviral vector method

Integrates the transgene into recipient cell genome

Disadvantage of the retroviral vector method

Only small pieces of DNA can be transferred

DNA microinjection method

Preferred method for producing transgenic mice

First step of DNA microinjection

Female mouse is stimulated to superovulate (~35 eggs)

What happens after superovulation

Females are mated and fertilized eggs collected

Where DNA is injected

Male pronucleus

Why male pronucleus is used

Larger and easier to locate

Type of DNA used

Linear DNA without prokaryotic vector sequences

What happens after injection

Eggs implanted into pseudopregnant foster mother

Time until birth

About 3 weeks

How transgene is detected

PCR or Southern blot

How DNA integrates

Randomly into genome

Embryonic stem cell method

Uses pluripotent cells from blastocyst stage embryo

Pluripotent

Can differentiate into all cell types

Why ES cells are useful

Can be genetically manipulated without losing pluripotency

Step 1 ES method

Create vector with transgene and homologous sequences

Step 2 ES method

Isolate ES cells from inner cell mass

Step 3 ES method

Transfect ES cells with recombinant vector

Integration method (ES cells)

Homologous recombination

Integration requirement

Must not disrupt essential genes

Positive selection

Cells survive G-418 due to Neoᵣ

Negative selection

Cells die in ganciclovir if incorrect insertion

Final ES steps

Inject into blastocyst and implant into foster mother

Knockout mice

Mice with gene inactivated to study function

Cre-loxP system

Controls gene expression in specific tissues

Problem with transgenic animals

Transgene expressed in every cell

Cre gene

Codes for recombinase

Cre recombinase function

Recombines DNA at loxP sites

loxP sites

Specific DNA sequences for recombination

Step 1 Cre-loxP

Cre under cell-specific promoter

Step 2 Cre-loxP

LoxP sites inserted into genome

Step 3 Cre-loxP

Cross two transgenic lines

Result Cre-loxP

Gene activated or inactivated in specific tissue

Cloning by nuclear transfer

Transfer nucleus into enucleated oocyte

Dolly the sheep

First cloned mammal from adult cell

Significance of Dolly

Adult cell nucleus is pluripotent

Step 1 cloning

Donor cells isolated and cultured

Step 2 cloning

Nucleus fused with enucleated oocyte

Step 3 cloning

Develops into blastocyst

Step 4 cloning

Implanted into pseudopregnant female

Step 5 cloning

Cloned animal is born

Cloning issue

Very low efficiency

Possible cloning problem

Telomere abnormalities

Plant biotechnology goal

Improve productivity, yield, and nutrition

Traditional plant methods

Crossbreeding and hybridization

Plant transgenesis

Direct transfer of genes into plants

Benefits of transgenic plants

Pesticide production, herbicide resistance, pathogen resistance, stress tolerance, improved nutrition, longer shelf life

Plant vaccines

Plants engineered to produce vaccine proteins

Three reasons for transgenic plants

Improve value, act as bioreactors, study gene function

Plant cloning

Growing plant from single cell

Unique plant cell feature

Can regenerate into whole plant

Callus

Mass of undifferentiated cells

Why callus useful

Can grow into whole plant and be manipulated

Cellulase

Enzyme that removes cell wall

Protoplast

Plant cell without cell wall

Protoplast fusion

Fusion of cells from different species

Leaf fragment technique

Main method using Agrobacter

Agrobacter

Soil bacterium that transfers DNA

T-DNA

DNA inserted into plant genome

Ti plasmid

Tumor-inducing plasmid

Natural effect of T-DNA

Causes crown gall tumors

Purpose of Ti plasmid

Vector for gene insertion

Ti plasmid requirements

Selectable marker, origin of replication, vir genes, cloning site

Selectable marker

Identifies transformed cells

Origin of replication

Allows plasmid replication

vir genes

Enable DNA integration

Cloning site

Where gene of interest inserted

DNA introduction into Agrobacter

Electroporation or calcium chloride method

Cloning vector

Carries gene of interest

Helper plasmid

Contains vir genes

Agrobacter infection

Enters through plant wounds

What activates vir genes

Chemicals from wounded tissue

Result of vir genes

Gene integrates into plant genome

Plant regeneration

Shoots from cytokinin, roots from auxin

Agrobacter limitation

Works only on dicots

Gene gun method

Used for resistant plants

Particles used

Gold particles

Gas used

High-pressure helium

After entry

DNA integrates into genome

Gene gun limitation

Large DNA may fragment

Chloroplast engineering

Inserting genes into chloroplast genome

Method 1

Gene gun delivery

Method 2

Fusion protein targeting

Two-plasmid system

Marker plasmid + gene plasmid

Integration method

Homologous recombination

Selection method

Antibiotic selection

Advantages chloroplast engineering

Multiple genes, high expression, not in pollen