April 24, 2003- Lecture Notes Outline

Topics: Transgenic Eggs and Animals

 

Applications: Why would one want to create transgenic animals?

1. Recombinant Protein Production

2. Gene Therapy

3. Reverse Genetics- introduce a specific change in the DNA, then ascertain the phenotype

4. Transient expression system

5. Genetically modified food production

6. Cloned people or endangered species

Xenopus oocytes

A. Arrested in 1st Meiotic prophase

B. Obtained from excised ovary

C. Use for microinjection and short term expression ( no stable lines)

1. DNA into germinal vesicle (nucleus)

2. RNA into cytoplasm

a. mixture of isolated mRNAs

b. in vitro transcribed RNAs (capped) either individual clone or a cDNA library

c. RNA is translated immediately, not good for studying events that occur past the mid-blastula transition

D. Protein products

1. Often correctly modified

2. Membrane proteins are often targeted appropriately

a. proven useful in cloning ion channels & receptors

b. requires that oocyte proteins do not interfere with protein function and that an assay is possible

E. Fertilized eggs

1. Injection of mRNA allows determination of a particular function during development.

2. Injection of DNA does not integrate into the genome and may be inherited in a mosaic pattern. There is also the limitation that many Xenopus promoters lack tissue specificity when not integrated into the genome.

Xenopus transgenics

A. Restrction enzyme mediated integration. The goal is to integrate a transgene into the genome of isolated sperm nuclei

1. Isolated sperm nuclei are incubated with linearized plasmid DNA

2. Add high speed interphase egg extract and a small amount of restriction enzyme used for plasmid linearization. This partially decondenses sperm chromatin but does not promote replication.

3. Incubate for a short time, then dilute the mixture 50-100 fold.

4. Transplant one nucleus /unfertilized egg (i.e. inject a small volume of the diluted solution which could contain zero, one or many nuclei).

5. Monitor the embryos for proper development.

6. Each animal is used as an individual experimental unit. Growing and mating genetically altered strains is usually not pursued due to the long life cycle of X. laevis

B. 20-50% of the injected eggs cleave and develop normally. Typically 5-40% of the cleaving eggs develop nomally beyond feeding tadpole stage, and the integrated DNA I expressed usually in a non-mosaic fashion.

Drosophila

A. P elements are present in many strains of Drosophila melanogaster.

a. some are defective - lack transposase necessary to catalyze P element mobility. Defective P elements can be mobilized by non-defective ones.

b. Autonomous P-element are 2907 pb, 20-50 copies/ genome have precise inverted repeats of 31 bp at the teminus.

c. engineered P-elements contain a selectable marker- usually eye color, or body color or G418 selection is possible. Other elements such as GFP, b-galactosidase, are incorporated as well to generate conditional or mosiacs.

 

B. Applications

a. Introduce gene stablily into the genome via p element mediated transformation

b. Allow non-specific integration- random placement- can't target genes for replacement

 

C. Procedures

Injection into the posterior of embryo prior to pole cell formation. Early development as a synchium so DNA is incorporation into the cell when membranes form.

Go- injected embryos, some of the DNA enters the pole plasm and is transcribed to produce transposase, transposase enters nuclei of germline cells and promotes transposition of defective transposon into the chromosome. Usually backcrossed with a stock line with recessive features to select for recombinant progeny.

F1- Transgenic progeny screened for visible markers

Enhancer Trapping

Remobilization to mutate genome.

Mice

A. Mini-gene Tansgenics

1976- R Jaenisch showed that MMLV could integrate into mouse chromosomes inheritably

1981- Brinster et al- demonstrate transgenic expression

Standard transgenics via pronuclear microinjection

Transgene is an expression cassette. Requires cis DNA elements to regulate proper gene expression (for beta globin gene full expression requires an enhancer 50 kb upstream of the transcriptional start. It is therfore important to realize that the absence of specific enhancer elements can significantly affect the pattern of transgene expression

Chromosomal integration usually occurs at 1 cell stage

20% transgenic will be mosaic

Concatamers are most common. Abnormal events can occur at the site of integration, such as deletions and/or rearrangements w/in the concatamer, or chromosomal deletions, duplications or translocations.

The concatamer (tansgene) may be unstable and may be lost after several generations

Contaminating DNA (unlinked) can be a problem. Avoid incorporation of prokaryotic sequences. Usually the transgene is excised from th cloning vector w/ restriction enzymes and is purified after separation on an agarose gel.

Detection of transgene-

Genotype using tail clippings. Isolate genomic DNA with proteinase K/SDS protocol. PCR for the presence of the transgene. Southern blot for copy number and or test for homozygosity. Stable integration occurs in 10-40% of the injected embryos.

B. Homologous Recombination (Gene Targeting)

1. Goal- to replace endogenous gene w/ sequences from the targeting vector

2. Replacement vectors- - replace

and insertion vectors - just insert in

3. Targeting vector has two arms

1 arm is shorter and is small enough to PCR screen for cells w/ the targeted mutations

Southern Blot analysis- 1-2 kb in length

Selectable marker such as neomycin resistance is present between the arms