BIO4320 Lecture Materials, Prepared by Dr.

Hon-Ming Lam

Transgenic Animals
Further Readings: Recombinant DNA (2nd ed.) by Watson et al. Principle of Gene Manipulation (4th ed.) by Old and Primrose Human Molecular Genetics (2nd ed.) by Sudbery, Ch.7

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

General Requirements and Considerations

Cells/cell line (a population of animal cells that develops on repeated secondary cultivations for an indefinite time) DNA of interest Selectable markers and concept of cotransformation Vectors and methods for transformation Transient versus stable (integration, genetargeting); making proteins versus studying of gene expression

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Selectable Markers
Enzyme (abbreviation) Aminoglycoside phosphotransferase (APH) Dihydrofolate reductase (DHFR): Mtx-resistant variant Drug for selection G418 (inhibits protein synthesis) Methotrexate (Mtx; inhibits DHFR) Selection mechanism APH inactivates G418 Variant DHFR resistant to Mtx HPH inactivates hygromycin-B TK synthesizes thymidylate

Hygromycin-B-phosphotransferase Hygromycin-B (inhibits (HPH) protein synthesis) Thymidine kinase (TK) Aminopterin (inhibits de novo purine and thymidylate synthesis) Mycophenolic acid (inhibits de novo GMP synthesis) 9--D-xylofuranosyl adenine (Xyl-A; damages DNA)

Xanthine-guanine phosphoribosyltransferase (XGPRT) Adenosine deaminase (ADA)

XGPRT synthesizes GMO from xanthine ADA inactivates Xyl-A

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Selection on HAT Media

Synthesis of purine nucleotides 5-phosphoribosyl-1-pyrophosphate blocked by aminopterin Uridine monophosphate blocked by aminopterin
Guanine monophosphate Adenine monophosphate Inosine monophosphate HPRT

Synthesis of thymidylate De novo synthesis

blocked by aminopterin Thymidine monophosphate thymidine kinase (TK) thymidine

hypoxanthine

Salvage pathway

DNA fragment from HSV

Target gene (e.g. rabbit -globin gene)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

tk gene

k k 4.7kb Calcium phosphate precipitation

Concept of Co-Transfection

Add DNA to tkcells

Add HAT medium Only tk+ cells grow Culture three tk+ colonies

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam Only tk+ cells grow Culture three tk+ colonies

Concept of Co-Transfection

Intetarted -globin genes

HSV DNA

Cellular DNA (chromosomal)

Cleave DNA with appropriate restriction enzymes (e.g. KpnI) Electrophoresis on agarose gel Southern blot hybridization with 4.7kb -globin probe DNA from colonies 1 and 3 contains -globin DNA

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Methods for Animal Gene Transfer

Naked DNA Microinjection, calcium phosphate precipitation, electroporation Lipoplexes Virus-mediated transfection E.g. Baculovirus (insect cells), retrovirus (human cells)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Microinjection
Size Extraction fractionation Affinity chromatography Poly(A)+RNA microinjection microinjection Poly(A)+RNA Assay for functional expression cDNA library synthesis In vitro transcription and capping Xenopus oocyte tissue

microinjection

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Successful Examples of Functional Expression in Xenopus Oocytes

Protein
Receptors Thyrotropin-releasing hormone receptor (mouse pituitary) Serotonin 5-HTIC receptor (mouse choroid plexus) Substance K receptor (bovine stomach) Glutamate receptor (rat brain) Platelet-activating factor receptor (guinea-pig lung) Carriers Na+/glucose co-transporter (rabbit intestine) Na+-independent neutral amino acid transporter (rat kidney) Channels K+ channel (rat brain) Frech et. al. 1989 Cl- channel (Madin Darby canine kidney epithelial cell) Paulmichl et. al. 1992 Hediger et. al. 1987 Tate et. al. 1992 Straub et. al. 1990 Lubbert et. al. 1987 Masu et. al. 1987 Hallmann et. al. 1989 Honda et. al. 1991

Reference

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Calcium Phosphate Precipitation

Using adenovirus DNA particles as an example Phosphate buffer Adenoviru s DNA

Add CaCl2

DNA precipitates Add DNA to cells

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

4-16hr 37C

Calcium Phosphate Precipitation

add fresh medium to cells 7 days 37C

Remove DNA solution

Transfer medium

Infectious viruses in medium

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Culture cells

Electroporation
DNA of interest Selectable gene

Mix DNA and cells in a special cuvette

Apply voltage

Cells uptake DNA through holes in membrane

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Apply voltage

Cells uptake DNA through holes in membrane

DNA enters nucleus

Apply selection Stable expression

No selection Transient expression

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Liposome-Mediated Gene Transfer

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Liposome adheres to cell Liposome bilayer fuses with plasma membrane DNA enters cell

Add to cells

LiposomeMediated Gene Transfer

Foreign gene expressed

DNA in nucleus

Foreign Protein Production in Insect Cells Using the Baculovirus Vector

Transfect insect cells by calcium phosphate method Cells take up plasmid and baculovirus DNA Culture cells

Foreign Protein Production in Insect Cells Using the Baculovirus Vector

Cells take up plasmid and baculovirus DNA Culture cells

Virus causes cells to lyse

Infect cells with recombinant virus Plaques (recombinant baculovirus will give plagues of different morphologies)

Harvest cells and assay for production of foreign protein

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Stable Transformation by Retrovirus

Transfect a packaging cell line Foreign gene LTR LTR

packaging

Retroviral vector DNA Isolate virus particles Cells express viral proteins Vector RNA is packaged into infectious virus particles Vector RNA

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Vector RNA Infect cells of interest Most cells are infected Expression of foreign gene

Stable Transformation by Retrovirus

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Common Techniques in Making Transgenic Animals

Mate mice

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

sperm eggs pronuclei of fertilized egg gene of interest pseudopregnant mouse

Microinjection into Pronucleus of a Fertilized Egg

Wash fertilized eggs out of oviducts Microinject foreign DNA into pronucleus

Suction pipette holding egg Implant eggs into pseudopregnant mouse Presence of transgene shown by PCR fragment

offspring DNA analysis

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Checking for Stable Integration

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Common Techniques in Making Transgenic Animals

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Mate mice and isolate blastocyst blastocyst Transfer to petri dish

Transformation of Embryonic Stem Cells

ES cells must be prevented from differentiating by growing them on a feeder layer of fibroblasts, or by adding leukemia inhibitory factor to the culture medium.

Plate out in petri dish with a feeder layer of cells

Remove inner cell mass and dissociate with trypsin Feeder layer

Transformation of Embryonic Stem Cells

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

ES cells must be prevented from differentiating by growing them on a feeder layer of fibroblasts, or by adding leukemia inhibitory factor to the culture medium.
DNA Transformation/Transfection
Feeder layer Plate without feeder layer Inject into blastocyst

muscle nerve

cartilage melanoblast Continue to culture feeder layer Chimeric mouse

Cell differentiation

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Cloned Animals

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Donor

Recipient

Somatic Cells

Ovum Removal of nucleus Cell Fusion

Pseudopregnant

Cell implanted into surrogated mother Surrogated mother gave birth to the progeny

Progeny (Genotype same as donor)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Potential Application of Transgenic Mammals

Gene expression studies (reporter genes, gene traps, etc.) Tissue/cell-type targeted gene expression (suicide gene, etc.) Gene targeted mutations (antisense, homologous recombination, etc.) Gene addition or replacement (e.g. gene therapy)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Reporter Genes (Gene Traps)

Gene trap construct Construct integrates within host gene

On activation a spliced transcript is produced

on translation, a gene XlacZ fusion protein and neo protein are produced

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Use electroporation to introduce construct into ES cells

Add G418 to select neo-containing cells Make a replica and stain with X-gal Colonies of surviving cells must contain neo gene

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Isolate colonies Inactive lacZ Grow cell lines Active lacZ Inject cells Examine embryos stained with X-gal No activation Activation during development Inactivation during development Regulated Expression expression throughout embryo

Genes regulated by development

Strong promoter Transfect cells tk gene for selection

Gene X cloned in opposite orientation to cellular gene X Antisense vector

Antisense vector Antisense RNA

Select tk+ cells

Cells lack protein X

Antisense Technology

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Double Selection for Homologous Recombination

Double Selection for Homologous Recombination

Ganciclovir is an antiviral agent that interferes with DNA synthesis. The drug is phosphorylated by viral thymidine kinases, and undergoes further phosphorylation by cellular kinases. In its triphosphorylated form, the drug inhibits DNA polymerase by acting as a terminator in the synthesis of DNA, and can induce apoptosis in cells infected by viruses.

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Gene Therapy
Somatic gene therapy: genetic defect is corrected only in the somatic cells of a person affected by the disease. Germline gene therapy: a genetic modification is made to a gamete, fertilized egg or embryo; these cells will develop into the whole body.

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Gene Therapy
Ex vivo gene therapy: introduce the transgene into isolate cells in vitro, followed by reintroduction of these cells into the patients body. In vivo gene therapy: introduce the transgene directly into the cells affected by the disease.

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

In Vivo Gene Therapy

Expressible remedial gene

Remedial gene construct

Ex Vivo Gene Therapy

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Adenosine Deaminase Deficiency (SCID)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Gene Therapy for Brain Tumors

Inject brain tumor with engineered retrovirus containing TK gene Treat with ganciclovir

A few cells are modified

The tumor is directly injected with a retrovirus containing the mouse thymidine kinase (TK) gene and a few cells take up the vector. These cells convert the prodrug ganciclovir into an active from and are killed. Because of the bystander effect surrounding cells are also killed.

Modified cells and bystanders killed. Healthy cells away from the tumor are not affected

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Will You Accept Somatic Gene Therapy?

Is it just in principle similar to organ transplant, blood donation, or other medical treatments? Counter argument: due to so much unknown in human genome and its regulation, if a treated patient turns out to be dangerous to the health of the society, could we just simply terminate the experimental target? People are dying (patient dont have time) versus precautionary principle (whats the rush, its not ready) How would you choose if your loved one is suffering? Where is the source of embryonic stem cells? Dead fetus? Abortion Fetus?