Transgenics Flashcards
(42 cards)
What is an ES cell?
An embryonic stem cell
What are the uses of ES cell chimeras?
Knockouts
Knockins
Genetraps
Conditional gene regulation
ES cells are pluripotent, what does this mean?
They contribute to many adult tissues including the germline
How can ES cells be transferred back to the embryo?
Microinjection
Aggregation
Outline how you would identify ES cells that contain homologous recombination events
- Generate targetting vector
- Transfect vector into ES cells
- Apply selection
- Pick individual colonies: freeze 80% of colony and prepare DNA from remaining 20%
- Screen for homologous recombination events by Southern blot analysis or PCR
What are the two ways genes can be targeted in ES cells?
Sequence replacement
Sequence insertion
Who derived the first mouse ES cells from a blastocyst and cultured them in vitro in 1981?
Evans and Kaufman
Martin
Who cultured the first embryos from and 8 cell blastocyst in 1949?
John Hammond
Who defined the exact conditions of in vitro culture of 1 cell blastocyst embryos in 1971?
Whitten and Biggers
What are the uses of direct DNA transgenics?
Lineage-specific expression
Reporter transgenes
shRNA
Large fragments of DNA: BAC/PAC (expression of multigene clusters)
Give an example of an application of lineage-specific expression in direct DNA transgenics
Melanoma development in mice expressing oncogenic G12D Ras from the tyrosinase promoter
Give an example of an application of reporter gene expression in direct DNA transgenics
Expression of lacZ gene in limb buds
Give an example of an application of shRNA inhibition of gene function in direct DNA transgenics
Inhibition of RAS.GAP generates defects in embryonic and extraembryonic development
Kunath et al., 2003
What are the three transgenic approaches?
Direct DNA transgenics
Embryonic stem (ES) cell chimaeras
Infection of embryos/ES cells with viruses
Outline how direct DNA transgenics is carried out
- Microinjection of vector into 1dpc mouse oocytes
2. Random integration of DNA into host genome
Outline how ES cell chimaeras are carried out
- Gene targeting in ES cells
- Targeted integration of vector into gene locus
- Microinjection/aggregation of ES cells into 2.5-3.5dpc mouse oocytes
Outline how the infection of embryos/ES cells with viruses is carried out
- Co-incubation of virus with ES cells or embryos
2. Random integration of virus into host genome
What is the infection of embryos/ES cells with viruses used for?
shRNA
Gene expression studies
What is talin?
A protein that mediates the attachement of cells to the extracellular matrix and is critical for cell migration
Give an example of an application of gene knockout in transgenics
Talin knockouts die in embryogenesis at E7.5 due to failure of mesodermal cell migration in gastrulation
Give an example of an application of gene traps in transgenics
Expression of myosin light chain 2v specifically in the embryonic heart
Describe conventional gene deletion
Deleted in all cells
Therefore, if embryonic lethal, cannot derive cell lines or study function in adult tissues
Describe conditional gene deletion
Circumvents embryonic lethality
Therefore can delete gene in any tissue and at any time in development or adult
Can derive cell lines for in vitro studies
What systems are used in conditional gene deletion?
Recombinase-based systems, e.g. Cre-lox, FRT-FLP
