Biomedical Engineering Reference
In-Depth Information
have been altered through the introduction of exogenous genes which promote homologous
recombination, including a defective
λ
prophage. The phage genes of interest,
exo
,
bet
and
gam
, are transcribed from the
λP
L promoter. This promoter is temperature sensitive and is
repressed at 32
◦
C and de-repressed at 42
◦
C. When bacteria are grown at 32
◦
C, no recom-
bination proteins are produced. However, briefly culturing the bacteria at 42
◦
C induces
expression of all the recombination proteins, which readily promotes the modification of
targeted DNA sequences.
These protocols allow the retrieval of a large region of mouse DNA contained in a BAC.
The retrieved sequence serves as the LHA for recombination in ES cells (see Figure 10.1 and
Figure 10.2). Recombineering bacterial strains can be obtained from The National Cancer
Institute at Frederick (NCI-Frederick).
In Ensembl, selection of desired BACs containing DNA from the 129 mouse strain
can easily be identified using the mouse genome browser (http://www.ensembl.org/
Mus_musculus/index.html). Here, 129 BACs are used in order to maximize recombination
efficiency in the R1 line of ES cells, which has a 129 genetic background. The first step
involves searching for the gene or region of interest and selecting the 'graphical view'
window. The region of interest should be visible with several other layers of information.
By default, BAC maps are not displayed, and in order to have the browser layer them
on the 'graphical View' it is necessary to select the 'DAS sources' menu and highlight
'
129S7/AB2.2 clones
'andthe'
BAC map
' option to view the span and genomic region
mapped to each library. Each individual BAC can be easily identified by its specific
library identifier and can then ordered online from Geneservice, Ltd, Cambridge, UK
(http://www.geneservice.co.uk/products/sanger/order.jsp) (bMQ 129 clones).
PROTOCOL 10.1
BAC Purification Protocol
Equipment and reagents
•
Luria-Bertani (LB) media with appropriate antibiotic resistance
•
37
◦
C incubator
•
Reagents included in the plasmid DNA purification Mini Kit (Qiagen)
•
2ml Eppendorf tubes
•
Isopropanol (Sigma)
•
70% ethanol
•
Microcentrifuge at 4
◦
C
•
15ml tubes.
Method
1 Grow bacteria containing the desired BAC in 6 ml of LB broth with appropriate antibiotic
resistance overnight at 37
◦
C with shaking (220 rpm).
2 Divide bacteria into three tubes and pellet by centrifugation at maximum speed
(16 000
g
)for1min.
3 Resuspend each pellet in 250
μ
l of buffer P1.
