Pronuclear microinjection of transgene constructs to create transgenic mouse lines.

 

MOUSE DNA TRANSGENES INJECTIONS

a)    The quality of the DNA preparation used in microinjection has a direct effect on the efficiency of DNA integration.

 

Each of the following parameters will affect the efficiency of DNA integration:

 

 

i) The injected fragment must be free of flanking plasmid sequences (which may be toxic).  Therefore, the construct should be designed so that the insert for microinjection can be excised without vector sequences.

ii) The DNA must be intact, without accompanying degradation, in order to prevent integration of partial constructs.

iii) The DNA must be free of contaminants such as phenol, ethanol, enzymes etc., which may harm the embryo.

iv) Solutions used in DNA purification and dilution buffers should utilize fresh, high quality sterile reagents, and Water for Embryo Transfer (Sigma W1503) must be used.

v) It is essential that the DNA solutions not contain any particulate material that may clog the microinjection needles.

vi) DNA concentration must be measured precisely in order for the final concentration for injection (2 ng/ul) to be accurately determined. The final dilution of DNA in injection buffer will be performed by the transgenic facility personnel.

vii) 50 ul purified DNA at a high concentration (50-100 ug/ml) should be delivered to the transgenic lab ten days before the assigned microinjection session, to check for DNA purity.

viii) It is highly recommended that you prove that your construct is expressed in vitro by an appropriate cell line. If this has not been shown for your construct, state it in your application.

 

b)    DNA will be injected into one of the pronuclei of the zygote of C57Bl6 background mouse and transferred to the pseudo pregnant female mouse.

 

c)    After 3 weeks weaning, pups will be delivered to the client for genotyping