Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.
For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.
The pioneering work of Gordon et al. in 1980 showed that after implantation in pseudopregnant mice, the plasmid DNA injection into the male pronuclei of fertilized oocytes can yield the production of transgenic animals that integrated the plasmid DNA1. The demonstration that transgenic mammals can be generated had an enormous impact on global life sciences, opening the way to novel fields of research both for basic sciences and translational biomedical sciences. In the past four decades, DNA microinjection has become a routine practice. Although an enormous number of transgenic mice have been produced, the standard method is not fully usable for all mouse strains and requires time consuming backcrosses2,3. Its application to other species remains challenging4 and the overall transgene integration yield is limited to a few percentage of born animals5. In addition, the efficacy of transgene integration represents the limiting factor that explains the poor overall yield of pronuclear DNA injection. In this respect, integrative viral vectors are the most efficient tools to cargo and integrate transgenes and thus could provide new means to significantly increase integration yield, the only limitation being that the transgene size that cannot exceed 10 kb6.
Lentiviral vectors pseudo-typed with the envelop protein of the Vesicular Stomatitis Virus (VSV) are pantropic and highly integrative gene transfer tools and can be used to transduce fertilized oocytes7. The zona pellucida surrounding the oocytes is a natural virus barrier and needs to be passed to allow transduction with the lentiviral vectors. Transgenic animals have been generated by transducing fertilized oocytes after micro-drilling or removing of the zona pellucida8,9. However, injection beneath the zona pellucida in the perivitelline space appears to be the simplest method to transduce the fertilized eggs as initially described by Lois and colleagues7.
The perivitelline injection of lentiviral vectors allows high yields in the production of transgenic animals that are above 70% of born animals. Such yield is over 10-fold higher than the best yield that can be achieved using standard pronuclei DNA injection7,10,11. In this context, a single session of injections will generate at least 50 transgenic founders (F0). The large number of founders is, therefore, compatible with phenotyping of the transgene effect directly performed on F0 mice without the need to generate transgenic mouse lines. This advantage allows for rapid screening of the transgene effect and is specifically adapted to perform in vivo gain and loss of function studies within weeks. In addition, regulatory DNA elements can also be rapidly screened to map enhancers and DNA motifs bound by transcription factors11,12. With pronuclear injections, transgenes usually integrate as multiple copies in a unique locus. With lentiviral vectors, integration occurs in multiple loci as a single copy per locus10,13. Therefore, the multiplicity of integrated loci is most likely associated to the very high expression penetrance observed in the transgenic founders, which makes the new generated model more robust.
Importantly, when using pronuclear injection of DNA, visualization of pronuclei during the procedure is absolutely required. This technical limitation prevents the usage of fertilized oocytes originating from a large variety of mouse strains. Therefore, production of a transgenic model in a specific strain for which pronuclei are invisible requires the production of animals in a permissive strain followed by at least 10 successive backcrosses to transfer the transgene in the desired mouse strain. With the lentiviral vector injections, perivitelline space is always visible and the injection does not require highly specific skills. As an example, NOD/SCID transgenic mice that are not appropriate for pronuclei injection have been obtained with the viral vector injections14.
Here, a comprehensive protocol is presented to allow simple production of transgenic mice using lentiviral vector injections in the perivitelline space of a one cell stage embryo. Transgene expression controlled with either ubiquitous or cell specific promoters is described in detail.
The pTrip ΔU3 lentiviral backbone was used in this study15. This vector allows for producing replication defective lentiviral vectors in which the U3 sequence has been partially deleted to remove U3 promoter activity and generate a self-inactivating vector (SIN)16. Lentiviral vector stocks were produced by transient transfection of HEK-293T cells with the p8.91 encapsulation plasmid (ΔVpr ΔVif ΔVpu ΔNef)6, the pHCMV-G encoding the vesicular stomatitis virus (VSV) glycoprotein-G17, and the pTRIP ΔU3 recombinant vector. The detailed production procedure is provided as supplemental methods.
Production of high titer lentiviral vector stocks is performed under Biosafety Level II conditions (BSL-2). This is true for most transgenes except for oncogenes that have to be produced in BSL-3. Therefore, production in BSL-2 conditions for most cases is sufficient. In addition, the use and the production are usually disconnected for most national regulatory agencies dealing with genetically modified organisms (GMO). Limited amounts of replication incompetent SIN lentiviral vectors (below 2 µg of p24 capsid protein) can be used under BSL-1 conditions as described by the French GMO agency in agreement with the European Union recommendations.
All procedures that include animal work have obtained ethical approval and have been authorized by the French Ministry of Research and Education under number APAFIS#5094-20 16032916219274 v6 and 05311.02. The ICM animal facility PHENOPARC has been accredited by the French Ministry of Agriculture under the accreditation number B75 13 19. The overall protocol requires performing each procedure within a precise time frame that is summarized in in Figure 1.
1. Animal Purchase and Preparation of Basic Compounds
2. Superovulation of Female Donors
3. Prepare the B6CBAF1/jRj Pseudopregnant Females
4. Fertilized Eggs Collection
5. Making Injection Pipettes
6. Making Holding Pipettes
7. Preparation of Injection Pipette Containing the Lentiviral Vector
8. Micro-Injection
9. Transferring Embryos into B6CBAF1/JRj Pseudopregnant Females
10. Genotyping Transgenic Founders
11. Quantification of Transgene Copy Number
Transgenic animals were generated using the protocol presented here. Representative results both ubiquitous and cell type specific transgene expression are illustrated.
Constitutive expression of transgenes
Ubiquitous promoters are basic research tools to express transgenes in a sustained and efficient manner. Such promoters are used for a very large variety of application from in vitro cell transfection to in vivo transgenesis in small and large animals.
Lentiviral vectors were constructed to express the green fluorescent reporter gene (eGFP) under the control of either the cytomegalovirus (CMV) promoter or the composite promoter CAG based on the fusion of the chicken actin promoter and the CMV enhancer. Both lentiviral vectors were produced (supplemental methods) and the titer was determined in 293T cells as transduction units (TU) based on eGFP expression. Both lentiviral vector constructs were injected in the perivitelline space of fertilized oocytes at a concentration of 109 TU/mL and implanted in pseudo-pregnant female mice. Implanted embryos were next collected just before birth and genotyped by PCR to follow eGFP integration. 73% (n=22) and 83% (n=32) of collected embryos had integrated the transgene for the CMV and the CAG lentiviral construct, respectively (Table 1). Transgenic embryos were then sectioned and immuno-stained for eGFP. As illustrated in Figure 3, only scattered eGFP positive cells are observed with the CMV promoter (Figure 3, top panel) whereas all cells expressed GFP when the CAG promoter was used (Figure 3, middle and bottom panels).
With the CAG promoter, 96% of collected transgenic embryos ubiquitously expressed the eGFP transgene (Table 1). Although both promoters are ubiquitous, only the CAG promoter is able to drive robust expression of the transgene in all cells. Alternative ubiquitous promoters were used such as phosphoglycerate kinase (PGK) and ubiquitin-C promoters and yielded similar results as the ones obtained with the CAG promoter with lower expression levels of eGFP (data not shown).
In vivo mapping of regulatory genomic regions to test tissue specific control elements.
For a large number of applications, expression of transgenes in a cell specific manner in transgenic animals is required. In addition, generation of transgenic animals can be highly instrumental to screen the ability of putative regulatory genomic DNA fragments to control cell specific expression of a given gene. As an example, lentiviral mediated production of transgenic animals was used to map cell specific enhancers that control Neurogenin 3 (Neurog3) expression11. Neurog3 is a basis Helix-Loop-Helix (bHLH) transcription factor that controls the commitment of pancreatic progenitors towards the endocrine fate. In Neurog3 null mutant mice, no endocrine cells in the pancreas can differentiate19. A 2.2 kb DNA fragment localized between positions -5284 and -3061 relative to Neurog3 transcription start site was cloned into a lentiviral vector upstream a beta globin minimal promoter to drive expression of an eGFP reporter gene as previously described11. A control construct was similarly generated by cloning a 2.4 kb intergenic fragment localized on mouse chromosome 6 (chr6: 14237279-14239685 relative to mm9 mouse genome assembly) in the same lentiviral backbone. This genomic region is localized within a 1 mega-base long gene desert between Gpr85 and Ppp1r3a genes. High titer lentiviral vectors were next produced using both constructs and named Neurog3-enh-eGFP and Chr6-eGFP.
Both lentiviral vectors were constructed and produced (supplemental methods). Since no cells expressing Neurog3 were currently available, the TU titer could not be determined. Alternatively, the titer was measured as concentration of p24 capsid protein. The 2 vectors were injected in the perivitelline space of fertilized oocytes and implanted in pseudo-pregnant female mice. The implanted embryos were collected at embryonic day 14.5 (E14.5) as this developmental stage corresponds to the maximal expression of Neurog3 in the pancreas. Embryos were next genotyped to follow eGFP integration. 84% (n=47) and 71% (n=48) of collected embryos had integrated the transgene for Neurog3-enh-eGFP and Chr6-eGFP lentiviral constructs respectively (Table 1). For each embryo, the pancreatic bud was dissected and then sectioned to perform immunostaining. 92% of Neurog3-enh-eGFP transgenic embryos expressed eGFP in the pancreas as illustrated in Figure 4 top panel (representative immunostaining). Importantly, the vast majority of eGFP positive cells were also Neurog3 expressing cells (Figure 4) indicating that the 2.2 kb Neurog3 enhancer is able to restrict eGFP expression within the Neurog3 cell population. By opposition, none of the Chr6-eGFP embryos expressed eGFP (Figure 4 bottom panel and Table 1) in the pancreas or outside of the pancreas. In addition, no ectopic expression of eGFP was observed outside of the pancreas in Neurog3-enh-eGFP embryos11.
For the 4 experiments presented above, a precise quantitative description of each step of the procedure is presented in Table 1. This illustrates the global efficacy of the procedure. Indeed, when comparing the numbers of collected animals that integrated the transgene with the number injected fertilized eggs, the global yield of the procedure is 44% in average of. The same yield with a pronuclear DNA injection of a construct containing the Neurog3 enhancer fused to the beta-galactosidase reporter does not exceed 3.1%.
Transduction of fertilized oocytes with a lentiviral vector leads to transgene integration that can occur at multiple sites10,13. The relative number of transgene integration sites were evaluated using quantitative PCR on genomic DNA (Figure 5). Quantification of eGFP integration was determined by quantitative PCR (qPCR) and normalized to Cdx2 gene that is present at 2 copies per genome as described previously11. The average number of integration sites was 19.36 ± 2.468 (S.E.M.) and 9.537 ± 1.186 (S.E.M.) in embryos generated from Neurog3-enh-eGFP and Chr6-eGFP construct, respectively. Interestingly, the two lentiviral vectors used to produce these animals presented different viral titers. The concentration of p24 capsid protein were of 124 ng/µL for Neurog3-enh-eGFP vector and of 52 ng/µL for the control Chr6-eGFP vector. It is most likely that such titer difference will account for the significant difference observed in integration site numbers in both population of transgenic embryos (Figure 5). This suggests that the average number of integration sites obtained in a batch of founder transgenic mice could be modulated by using viral stocks with different titers.
Importantly, no direct correlation was observed between the expression of eGFP in Neurog3-enh-eGFP transgenic embryos and the number of transgene copies that were integrated. In other words, embryos that integrated either single or multiple copies of the Neurog3-enh-eGFP transgene were similarly found to express eGFP in Neurog3 positive cells.
Figure 1: Flow chart of the overall procedure Please click here to view a larger version of this figure.
Figure 2: Preparing the micro injection pipettes and genotyping.
(A) Schematic drawings of microinjection pipettes to highlight main differences between pipets used for DNA or lentiviral vector injections. Left panel: the overall shape of both pipette types is drawn. The dashed circle highlights the enlarged area of the pipet tip. Pictures of the pipet tips are also presented. Note that for lentiviral injection the tip needs to be broken as indicated with the dotted line and the corresponding picture. Right panel: example of egg injection setting with the holding pipet on the left, the fertilized egg and injection pipet either in one pronucleus or in the perivitelline space. Scale bars = 50 µm. (B) Visualization on agarose gel of eGFP PCR product amplified from genomic DNA extracted from 8 different embryos (lane 1 to 8). Only embryos 1, 2, 3, 5, 6 and 8 had integrated the eGFP transgene. The DNA plasmid pTrip PGK-eGFP used for lentiviral vector production was used as PCR positive control. For the negative control, H2O replaced DNA in the PCR reaction. MWM: molecular weight marker. bp = base pair. Please click here to view a larger version of this figure.
Figure 3: Ubiquitous promoters drive expression of the eGFP reporter in transgenic embryos.
10 µm cryo-sections of transgenic embryos were stained to visualize eGFP expression (green) and nuclei (blue). Embryos generated with the CMV promoter lentiviral construct (top left label) were collected at E11.5. Embryos generated with the CAG promoter lentiviral construct (bottom left label) were collected at E18.5. Pb: pancreatic bud, VSC: ventral spinal cord, Vt: vertebra, Li: liver, Ms: muscle of the abdominal belt. Scale bars = 50 µm Please click here to view a larger version of this figure.
Figure 4: Cell specific expression of reporter gene in transgenic embryos is driven by the Neurog3 enhancer. 10 µm cryo-sections of E14.5 pancreatic buds of transgenic embryos were stained to visualize the expression of Neurog3 (red), eGFP (green) and nuclei (blue) as described in supplemental methods). Neurog3 expression is scattered in the pancreas. Transgenic embryos that integrated the Neurog3-enh-eGFP construct express eGFP and most of eGFP positive cells are Neurog3 positive (top panel). Embryos generated with the Chr6-eGFP construct were not expressing eGFP (bottom panel). Scale bars = 50 µm Please click here to view a larger version of this figure.
Figure 5: Relative copy number of integrated transgenes. Quantification of eGFP integration sites relative to CDX2 gene as described in protocol section. Box plot from 25th to 75th percentile. Dots represent the different transgenic embryos that were generated. The comparison of transgenes integrated sites between the two lentiviral constructs is significantly different (Unpaired parametric t-test, p = 0.001). Please click here to view a larger version of this figure.
Table 1: Step by step quantitative report during the complete procedure. During the course of the procedure, the total numbers of eggs or embryos were counted. The first column represents the total number of eggs that were retrieved from the oviducts of superovulated females. Only eggs with clear 2 polar bodies and/or visible pronuclei were injected and are reported. After injection and a few hours in culture only the injected eggs that were not lysed and had a normal morphology were implanted. Next the total number of embryos that were collected from the pseudopregnant females are counted. Finally, embryos that had integrated the transgene and expressed the reporter are listed in the last two columns. The same features are also given for comparison with an experiment using standard pronuclei DNA injection. Here the transgene contained the Neurog3 enhancer driving expression of a beta-galactosidase reporter gene (Neurog3-enh-LacZ). Please click here to download this file.
The perivitelline injection of lentiviral vectors in fertilized oocytes described here resulted in the production of transgenic embryos that yielded more than 70% of transgenic embryos relative to total number of collected embryos. This result is consistent with previous reports and exemplifies the specificity of the procedure2,7,10,11,12. When comparing all the data presented in Table 1, important features can be highlighted. First, the number of implanted eggs corresponded to all injected eggs that had a normal morphology or were not lysed after few hours in culture. 93% of injected eggs were implanted suggesting an almost complete absence of rapid toxicity due to the injection of lentiviral vector in the perivitelline space. The situation is dramatically different when considering DNA injection since only 44% of injected eggs had survived and were implanted. Furthermore, the ratio of collected embryos relative to implanted eggs is identical between the two procedures, suggesting no exacerbated long-term toxicity of lentiviral vectors. Second, when expressing the number of embryos that integrated the transgene relative to the number of injected eggs the global yield is more than 10 times higher with lentiviral vector injection compared to DNA injection. An 86-fold difference is even found when comparing the numbers of embryos expressing transgene between the two procedures using the same Neurog3 enhancer construct.
Importantly, transgenic production yield appears to be dependent of the transduction titer of the used lentiviral vectors. In other words, lentiviral vectors produced with a titer above 109 TU/mL are sufficient to obtain such high yield. As described in the protocol section, the injected volume in the perivitelline space is in the range of 10 to 100 pL. This volume will represent 10 to 100 active lentiviral particles. In comparison to standard pronuclei DNA injections, the total number of founder animals generated with the same amount of born animals is at least 10-fold higher when using lentiviral vectors. In addition, the expression penetrance of the transgene is extremely high with this protocol and was observed both with ubiquitous and cell specific promoters with the exception of the CMV promoter. By opposition to cellular ubiquitous promoters, the CMV promoter is actively shut down by DNA methylation20 and has been shown to be unable to maintain long term expression upon transduction in pluripotent stem cells21. This could explain the very limited number of eGFP expressing cells observed in the transgenic embryos. Therefore, lentiviral vectors are well adapted to produce transgenic animals in which expression of a transgene is controlled by a cell specific enhancer. Importantly, the protocol can be used to screen for enhancer activity in vivo and to find map transcription factor binding sites within regulatory regions11,12. This screening approach can hardly be performed using standard transgenesis. The total number of founder animals needed to test all the different constructs and reach statistical significance would require dozens of injection sessions whereas it can be obtained rapidly with lentiviral mediated transgenesis.
One of the main differences between the standard procedure and the lentiviral based method resides in the transgene integration. Using pronuclear injection, transgenes integrate randomly as multiple copies in a unique locus. Using lentiviral vectors, integration can occur at multiple loci (one copy per locus) without being strictly random. By cloning integration sites using Linear Amplification Mediated PCR (LAM-PCR), the group of D. Trono has shown that transgenes integrate preferentially into open chromatin regions of the fertilized eggs13. The integration bias should not interfere or contribute to the transgene expression in the transgenic mice. Integration during lentiviral transduction in a one cell stage embryo occurs in open chromatin that may not be still in the open configuration later during development or in the adult.
In addition, when analyzing copy numbers of integrated transgenes in first generation animals (F0) or embryos, a large variation in number of integrated transgene is observed. In this study, an average of 19 integrated copies was found with the Neurog3-enh-eGFP construct. This large copy number could reflect high levels of mosaicism. Sauvain et al. have performed an extensive study of integrated loci in F0 animals generated with the lentiviral mediated method described here13. They followed 70 individual integration sites in 11 F0 animals and examined the rates of transmission for each site from F0 transgenic mice to their F1 progeny. The overall rate of transmission of 44% for individual integrated transgene suggests that they were most often established either after the S phase of one cell embryos or before the S phase at the two-cell stage. Indeed, integration prior to S phase would transmit the integrated transgene to both daughter cells, while integration after the S phase would transmit it to only one daughter cell. Thus, the degree of mosaicism for individual integrated transgenes is minimal in transgenic mice obtained through this technique. This further indicates that most integration will occur within the first 12 h corresponding to the average time of production of the first cleavage in the used culture conditions. This integration kinetic is consistent with the one described for lentiviruses in T-lymphoid cells22.
Importantly, with a high number of loci baring integrated transgenes, establishing mouse lines would not be reasonable. The number of crossings to segregate all these loci would be considerably high. This represents one important limitation of this method that should be used either for rapid screening of transgene effects or for simultaneous analysis of multiple transgenes. Nevertheless, mouse lines can still be established by selecting from the F0 animal the ones with the lowest integrated transgene copy number.
Since the first description of the pronuclear DNA injection method1, improvements have been made that circumvent many of the drawbacks of the initial procedure. The first set of improvements was based on the targeted integration in a precise locus using a cassette exchange strategy. Pronuclear injection is performed using either CRE, Flip or PhiC31 recombinases together with an integrative DNA fragment flanked with loxP, FRT or attB sites, respectively. In this situation, the integrative DNA is exchanged with an integrated fragment flanked with the same recombinase specific site23,24. Although up to 60% of first generation animals can be transgenic23 using such method, the limitations linked to the technology of pronuclear injection still apply. The second set of improvement is based on cytoplasmic injections of two circular DNA, one carrying the fragment to integrate and one allowing expression of either the Tol225, Sleeping Beauty26 or piggyBac27 transposases. Using these methods, high yields are obtained (>30%), but more importantly, the cytoplasmic injection is easy to perform and circumvents the restrictions due to pronuclear injection as the lentiviral based protocol. Furthermore, very large DNA fragments, such as bacterial artificial chromosomes, can be integrated.
It is clear that lentiviral mediated transgenesis will not replace the standard nor the improved procedures. Still this method represents a powerful tool for rapid animal model production and characterization as it considerably reduces the required time to generate proper number of animals with the least genetic variability. Furthermore, this technology can be directly applied to all mouse strains including any transgenic lines. In addition, it is crucial to mention that the global landscape of generation of novel animal models is about to change with the recent development of the CRISPR/Cas9 technology. Today, pronuclear injections of Cas9 protein along with guide RNA allows production of genome edited animal models with an efficacy of 40%28. This approach could largely benefit from the use of lentiviral mediated transgenesis. Indeed, the use of non-integrative lentiviral vectors29 to transiently express both Cas9 and guide RNAs could result in even higher production yields. The combination of the newest technologies to produce relevant and robust animal models would benefit most international research groups involved in studying disease pathogenesis and therapeutic approaches.
The authors have nothing to disclose.
We thank Magali Dumont and Rolando Meloni for critical reading of the manuscript and the iVector and Phenoparc ICM Cores for technical assistance in lentiviral vector production and animal housing respectively. This work was supported by the Institut Hospitalo-Universitaire de Neurosciences Translationnelles de Paris, IHU-A-ICM, Investissements d'Avenir ANR-10-IAIHU-06. P.R. received funding for the Association de Langue Française pour l'Etude du Diabète et des Maladies Métaboliques (ALFEDIAM) and a joint JDRF / INSERM grant.
PMSG 50UI | Sigma | G4527 | |
hCG 5000UI | Sigma | CG5-1VL | |
NaCl | Sigma | 7982 | |
100 mm petri dish | Dutsher | 353003 | |
4 wells Nunc dish | Dutsher | 56469 | IVF dish |
M2 medium | Sigma | M7167 | |
M16 medium | Sigma | M7292 | |
0,22 µm Syringe filter | Dutsher | 146611 | |
Hyaluronidase Enzyme 30mg | Sigma | H4272 | mouse embryo tested |
Insulin serynge | VWR | 613-3867 | Terumo Myjector |
Curved forceps | Moria | 2183 | |
Curved scissors | Moria | MC26 | |
Aspirator tube assemblies for calibrated microcapillary pipettes | Sigma | A5177-5EA | |
Borosilicate glass capillaries | Harvard apparatus | GC 100-10 | |
Horizontal micropipette puller | Narishige | PN-30 | |
Microforge | Narishige | MF-900 | |
Inverted microscope | Nikon | Transferman NK2 5188 | Hoffman modulation contrast illumination is required |
Micromanipulator | Eppendorf | Celltram air | |
Controler of holding pipet | Eppendorf | Femtojet | |
Mineral oil | Sigma | M8410 | mouse embryo tested |
Microinjector | Eppendorf | Femtojet | Can be used to inject DNA or viral vectors |
Dumont # 5 forceps | Moria | MC 40 | |
vannas micro scissors | Moria | 9600 | |
Isoflurane | centravet | ISO005 | ISO-VET 100% 250ml |
ocrygel | centravet | OCR002 | |
Povidone iodure | centravet | VET001 | vetedine 120ml |
Buprenorphine | centravet | BUP002 | Buprecare 0,3Mg/ml 10ml |
Tris-HCl | Sigma | T5941 | Trizma hydrochloride |
EDTA | Sigma | E9884 | |
SDS | Sigma | 436143 | |
NaCl | Sigma | S7653 | powder |
proteinase K | Sigma | P2308 | |
oneTaq kit | NEB | M0480L | |
Primers | Eurogentec | ||
Strip of 8 PCR tube | 4titude | 4ti-0781 | |
96 well thermal cycler | Applied Biosystems | 4375786 | Veriti |
Genomic DNA mini kit | invitrogen | K1820-02 | |
Nanodrop 2000 | Thermo Scientific | ND-2000C | |
qPCR Master mix | Roche | 4887352001 | SYBR Green |
Multiwell plate 384 | Roche | 5217555001 | |
qPCR instrument 384 well | Roche | 5015243001 | LightCycler 480 |