Authors: Subeer S. Majumdar , Suveera Dhup & Abu-ul Usmani
Presently used techniques for making transgenic animals are cumbersome; require trained man-power, costly infrastructure and large number of zygotes at the expense of several females. The ability of the male germ cells to integrate foreign genes provides opportunity for developing alternate methods for generation of transgenic animals. We have developed a reproducible protocol for transfecting mammalian and non-mammalian genes in repopulating undifferentiated spermatogonial cells through in vivo electroporation of the testis. More than 90% of the male mice electroporated with transgene of choice sire transgenic pups upon mating with the wild type (WT) females. Such males serve as permanent resource for the production of transgenic founders. This technique has several advantages over the presently used techniques including drastic reduction in the time required and in the usage of animals for generation of transgenic progeny. Hence, this protocol is likely to be applicable to many species.
Transgenic animals have become one of the most sought-after tools for the biological inventions. Pronuclear DNA microinjection in the oocyte is the frequently used technique for generating transgenic mice (1). This technique is not easy and has limited success rate. Other methods for generating transgenic animals, such as viral transduction or cloning are equally complicated and require co-ordination of a number of experimental steps (1). The complexities of the existing technologies and need of several donor females restrict their widespread use including that in large animals (2,3). With the availability of human, mouse and rhesus monkey genomes, use of transgenic animals in basic as well as in applied research is bound to increase. This generates an urgent need to develop an alternate, cost-effective, user friendly and more rapid approach for obtaining desired transgenic animals, preferably with minimum or no loss of animal lives. Here we present a protocol for generating transgenic mice by in vivo electroporation of the desired transgene in the testis. This protocol leads to permanent integration of the transgene in the repopulating spermatogonial cells. Thus, such males can continuously generate founder transgenic animals over a long period of time. The procedure of DNA injection in the testis and electroporation is accomplished in about 30 minutes. It neither requires large number of females for super ovulation nor requires surrogate mothers for embryo transfer. Hence, this protocol increases the probability of generating transgenic cattle and subhuman primates also. This technique is ethically superior as compared to presently prevailing techniques because it does not compromise with the lives of animals.
Any strain of mice can be used. Thirty days old males are most suitable for better outcome of this protocol with the advantage that a large proportion of the testicular germ cells can be easily accessed from the interstitial side of the testis in this age group of mice. Routinely, we use 28-32 days old mice (FVB/J) weighing about 20-25 g. The same protocol may be tried with minor modifications for animals of different age and species.
Plasmids having gene of interest cloned under an appropriate promoter which is functional in mammals.
DH5α strains of E. coli can be obtained from Stratagene (USA).
Media for growing bacteria
Tryptone, Yeast extract and Bacterial agar can be obtained from Pronadisa (Spain).
Plasmid isolation kits (both mini and maxi-scale) and Gel extraction kit can be obtained from QIAGEN, (Germany).
The appropriate restriction enzymes which cut the plasmid either at one site or at two sites should be used for obtaining the desired linearized fragment, containing promoter and gene of interest, for testicular injection and transfection. The size of DNA fragment for injection should ideally be less than 10 kb.
For the purpose of anesthesia, use a mixture of Ketamine hydrochloride and Xylazine hydrochloride; we use stock solutions of 50mg/ml and 20mg/ml respectively. Mix these solutions and dilute with normal saline to get a final concentration of 160µg of Xylazine hydrochloride and 900 µg of Ketamine hydrochloride per 100 µl.
Trypan blue Dye, Tris-HCl, Sodium dodecyl sulphate (SDS), Sodium Chloride (NaCl), EDTA, Proteinase K, Paraformaldehyde, Glycerol, Ampicillin (sodium salt) and Kanamycin monosulphate can be obtained from Sigma Chem. Co. (USA). dNTPs can be obtained from Promega (USA). Taq Polymerase and Molecular weight markers can be obtained from New England Biolabs (USA).
P-97 Horizontal pipette puller from Sutter Instruments Co., USA.
An electric pulse generator, Electroporator-ECM2001 from BTX Instrument Division, Harvard Apparatus, Inc., USA may be used for electroporation. A tweezer type electrode works better for holding and in vivo electroporation of the testis.
Peltier Thermal Cycler PTC-200 with heated lid (MJ Research, Minnesota, USA).
UV-2450, UV-VIS Spectrophotometer (Shimadzu Corp., Kyoto, Japan).
Maintenance of bacterial cultures and plasmid isolation
Agarose gel electrophoresis and extraction of DNA from the gel
Preparation of glass micropipette
In vivo electroporation of the desired gene in the testis
CAUTIONS All animal studies should be performed with relevant institutional guidelines, permissions and regulations. Handle animals with institutionally approved ethical procedures and avoid stress to the animals. All steps described below should be performed under sterile laminar flow hood.
Establishment of transgenic lines
Note: In mice, it takes about 30 days to complete a cycle of spermatogenesis during which period a spermatogonia differentiates into sperm. Hence, transgenic sperm produced after 30 days of electroporation presumably originates from the spermatogonial cells in which permanent integration of the transgene occurs at the time of electroporation.
Screening of transgenic pups using Polymerase Chain reaction (PCR)
The protocol presented here allows generation of transgenic mice within a short period of time. This deathless technique does not require highly trained manpower and expensive infrastructure, hence, can be used under standard laboratory conditions easily by junior and senior level bench workers. Using parameters described in this protocol, we generated transgenic mice using linearized IRES2-EGFP plasmid (Clontech, USA) which contains Cytomegalovirus immediate early promoter/enhancer (PCMV-IE) and the EGFP gene. The insert (linearized DNA) for testicular microinjection was generated by digesting the plasmid with Cla I. Transgenic animals were made using this New Spermatogonia Mediated (NSMED) technique. The PCR results of two generations of progeny are shown in Fig.2. In our hands, such electroporated males have sired transgenic pups even after 300 days of electroporation suggesting permanent integration of the gene in the repopulating spermatogonial cells at the time of electroporation. Reproducibility of this protocol has been reported by us using four different constructs and additional methods of screening viz. Southern blot, RT-PCR and Immuno-histochemical analysis. The success rate of this protocol is more than 90% which is better than the existing protocols used for the generation of transgenic mice.
This work was funded by the National Institute of Immunology and Department of Biotechnology, India.
Figure 1.: Electroporation of the gene in the testis
Table 1.: Various electroporation conditions used while standardizing in vivo testicular electroporation of the linearized gene fragment in mice.
Abbreviations used. DOE: date of electroporation, Conc.: concentration, Vol.: volume, Volt.: voltage, EP: electroporation, t: time constant, No.: number of pulses, F: forward direction, R: reverse direction. Highlighted row depicts the most suitable condition of electroporation.
Figure 2.: a. Details of the transgene used for electroporation. b. Determination of transgene (IRES2-EGFP ) by PCR.
Results of PCR using genomic DNA (gDNA) obtained from tail biopsies of progeny generated from fore-founder 13M electroporated with IRES2-EGFP. A large number of samples from different transgenic lines were run on the same gel. However, portions relevant to this figure were cut from the original gel pictures and shown here. Lanes 1-5:G32-36, Lanes 10-21:G56-68, Lanes 22-26:G89-93, Lanes 30-39:G139-148, Lanes 6,7,27,40 and 41 = plasmid DNA, Lanes 8,28, and 42 = gDNA of WT mice, Lanes 9,29 and 43 = blank without any DNA. G= prefix used to denote progeny of electroporated mice, WT = wild type mice, + = plasmid DNA, − = gDNA of WT mice, B = blank without any DNA. F1 = animals generated by mating of electroporated male with WT female. F2 = animals generated by mating of PCR positive male (G89) of F1 generation with WT female. Primer sequence for EGFP: Forward sequence = ACGTAAACGGCCACAAGTTC Reverse sequence = GGCGGTCACGAACTCCAG
Transgenesis via permanent-integration of genes in repopulating spermatogonial cells in-vivo, Suveera Dhup and Subeer S Majumdar, Nature Methods 5 (7) 601 - 603 15/06/2008 doi:10.1038/nmeth.1225
Subeer S. Majumdar, Suveera Dhup & Abu-ul Usmani, Division of Embryo Biotechnology, National Institute of Immunology, New Delhi- 110067, India.
Source: Protocol Exchange (2008) doi:10.1038/nprot.2008.124. Originally published online 19 June 2008.