The overall goal of this procedure is to produce mutant fish using gene breaking transpose on vectors with GAL four, VP 16 as the primary gene trap reporter. This is accomplished by first co injecting the gene trap vector along with tall two transposes into zebrafish embryos. Injected embryos are screened for GFP fluorescence at three days post fertilization and approximately 30%of the brightest embryos are selected to establish the F zero pool.
Next, the F zero fish are crossed to the U-A-S-M-R-F-P tester line to screen for germline transmission of gene trap events. The final step is to outcross the F1 to establish the F two generation and to generate embryos for molecular characterization of gene trap events by inverse PCR and five prime race. Ultimately about one in 10 screened F zero fish should produce gene trap progeny.
Gene breaking transpos were designed to produce no mutations upon integration into entrances of protein coding genes. Our GBT vectors use G four P 16 as the primary gene trap reporter. This modification increases sensitivity for genes expressed at low levels and makes it possible for us to use gene trap mutants without over avoid phenotypes as G four drivers for tissue specific expression of other transgenes.
We believe the main bottleneck of our method is in achieving sufficiently high rates of gene trapp integration in the germline of F zero fish. In this demonstration, we will cover several critical steps, DNA preparation, MICROINJECTION and F zero screening. The GBTB one gene trap vector uses GAL four VP 16 as the primary gene trap reporter.
Minimal tall two transpose on ends are used for gene trap integration. Gene trap events are directly detected by an EGFP reporter under the control of 14 X GAL four UAS. The whole gene trap cassette is flanked by direct locks P sites.
This makes gene trap events reversible by expression of Cree combs for establishing proof of a causality relationship between a specific gene trap integration and an observed phenotype. In addition, the U-A-S-E-G-F-P cassette is flanked by direct FRT sites. Injection of flip combs mRNA will lead to excision of the U-A-S-E-G-F-P cassette, leaving the gene trap mutation unmarked by EGFP fluorescence and enabling its use in transgenic lines that mark specific tissues or developmental events by GFP fluorescence prior to micro injection.
Prepare the transpose on DNA and transposes mRNA. Prepare the GBTB one gene trap vector DNA. Using a standard mini prep kit and following the manufacturer's protocol, it is essential to include the PB wash step when preparing the DNA.
Otherwise, the mini prep DNA will be contaminated by RNAs, A leading to degradation of the transposes Mr.NA, dilute the DNA in RNAs free water to 10 nanograms per microliter. To prepare the tall two transposes mRNA first linearized PT three TS T two using xbo one. Next, transcribe the Linearized DNA using an M message machine T three in vitro transcription kit with one modification.
Add 0.5 microliters of rib lock RNAs inhibitor after the DNA's treatment step purify mRNA using a standard kit and assess the quality of the in vitro transcribed mRNA by agros gel electrophoresis. Dilute the mRNA in RNAs free water to 40 nanograms per microliter and store as two microliter aliquots at negative 80 degrees Celsius just before the micro injection. Prepare the injection mix by adding eight microliters of diluted transpose on DNA to a two microliter aliquot of transposes mRNA.
Inject three nanoliters of D-N-A-R-N-A mixture into the yolk of one cell zebra fish embryos using standard microinjection techniques aiming for the yolk blasphemer interface After injection run five microliters of leftover injection solution on a 1%aros gel for quality control to ensure that the transposes mRNA has not been degraded about six to eight hours after injection. Remove unfertilized and dead embryos and distribute embryos to 60 to 80 per 100 millimeter Petri dish. Abnormal and dead embryos are removed at one day post fertilization, two days post fertilization and three days post fertilization at three days post fertilization under a stereo microscope equipped with fluorescence or an upright microscope.
Screen the embryos and remove any abnormal embryos. Screen the remaining embryos without overt defects. For GFP fluorescence, select the brightest F zero embryos and raise them using standard zebra zebrafish rearing procedures in this laboratory.
GBTs are usually injected into lines with wild type or leopard pigmentation patterns. It is reasonable to expect that 20 to 30%of the injected embryos selected for rearing will survive into adulthood. Integration of the GBTB one gene trap vector can lead to EGFP expression by two different mechanisms.
The first is a true gene trap event. The vector integrates into a gene a fusion transcript between the five prime of that endogenous gene transcript and the GAL four VP 16 is made and translated into a fusion protein containing the end terminus of the protein and coated by the insert mutated gene and GAL four, VP 16. This fusion protein binds to the 14 XUAS and activates transcription of EGFP.
The second less desirable event is an enhancer trap. The minimal promoter in front of EGFP falls under the control of an enhancer near the integration site leading to production of EGFP in the absence of GAL four, VP 16. To distinguish between these two classes of events, a 14 X-U-A-S-M-R-F-P transgenic line marked by lens specific gamma crystalline GFP was made.
Gene trap events are verified by crossing GBT injected fish with homozygous U-A-S-M-R-F-P fish and screening for co-expression of GFP and RFP, which is only possible if GAL four, VP 16 is made and activates U-A-S-M-R-F-P in trans to begin the procedure for screening F zero fish cross individual F zero with homozygous U-A-S-M-R-F-P fish. Since the GBT injected fish have wild type or leopard pigmentation patterns and the homozygous U-A-S-M-R-F-P line is in a brass background, F zero fish can be readily distinguished from the U-A-S-M-R-F-P fish eliminating the need to record whether the F zero being screened was a male or a female, as well as potential errors resulting from mistakes in recording and or determining the sex of each fish. On the next day.
Return the brass U-A-S-M-R-F-P fish to their tank. Place the F zero fish that gave embryos into individual static tanks while embryos are collected for screening clean and transfer collected embryos into new Petri dishes in the afternoon of day zero. Screen embryos for GFP and RFP fluorescence at one day post fertilization and three days post fertilization.
Pull out embryos positive for both GFP and RFP sort them by G-F-P-R-F-P expression pattern. If more than one pattern is observed in a clutch and raise them to establish the F1 generation. Note that 3D PF embryos that are positive for GFP but not for RFP are not pulled out as they represent enhancer trap events.
Cross the F zero fish which produced G-F-P-R-F-P positive progeny again to get a second batch of positives. The F1 fish are screened to establish F two families to document gene trapp expression pattern and to freeze batches of embryos for molecular identification of gene trap loci First cross individual F1 fish with the homozygous U-A-S-M-R-F-P fish on the following day. Place the F1 fish that gave embryos into individual static tanks while the embryos are collected for screening at one day post fertilization.
The RFP labeling may not have come up yet, so screen the embryos for GFP fluorescence and pull out the GFP positive embryos at three days post fertilization screen for both GFP and RFP fluorescence and pull out the double positives. Then photograph the embryos positive for both GFP and RFP at five days post fertilization freeze batches of 20 G-F-P-R-F-P positive and 20 G-F-P-R-F-P negative embryos for identification of insert mutated genes by inverse PCR and five prime race. Raise the remaining G-F-P-R-F-P positive embryos to establish the F two generation in a successful injection.
At least 80%of the embryos injected with the gene trap DNA tall two transposes mRNA mix will display some degree of GFP fluorescence. Low GFP expression indicates either unsuccessful injection or degradation of transposes RNA due to RNAs contamination. When the brightest GFP positive embryos are selected to establish the F zero pool, about one in 10 screened F zero fish will yield gene trap progeny among the F zero fish from which the gene trap events are recovered.
Most transmit a single gene trap event in addition to several non expressing transpose on integrations. Since the whole gene trap cassette is flanked by direct locks P sites, any gene trap mutations illustrated in this diagram with blue boxes representing exons of the mutated gene is reversible by expression of CRE combs. These images show the co-expression of GFP and RFP in the nervous system of the NSF TP six gene trap line injection of Cree RNA into N SF TP six embryos leads to loss of GFP and RFP expression.
Note that there is no reduction of GFP expression in the lens as expected. Most of the procedures outlined here are applicable to other gene trap screens as well as regular transgenesis experiments with TOL two and other transposons With the gene trap procedure outlined here. Setting up 20 to 30 F zero out crosses a week should result in the recovery of one gene trap per week.
However, the procedure can easily be scaled up.
