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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: J Mammary Gland Biol Neoplasia. 2020 Nov 9;25(4):389–396. doi: 10.1007/s10911-020-09469-w

Intraductal Injection of Lentivirus Vectors for Stably Introducing Genes into Rat Mammary Epithelial Cells in Vivo

Wen Bu 1,2, Yi Li 1,2,3,*
PMCID: PMC7965254  NIHMSID: NIHMS1645321  PMID: 33165800

Abstract

Various retroviral and lentiviral vectors have been used for up-the-teat intraductal injection to deliver markers, oncogenes, and other genes into mammary epithelial cells in mice. These methods along with the large number of genetically engineered mouse lines have greatly helped us learn normal breast development and tumorigenesis. Rats are also valuable models for studying human breast development and cancer. However, genetically engineered rats are still uncommon, and previous reports of intraductal injection of retroviral vectors into rats appear to be inefficient in generating mammary tumors. Here, we report, and describe the method for, stably introducing marker genes and oncogenes into mammary glands in rats using intraductal injection of commonly used lentiviral vectors. This method can infect mammary epithelial cells efficiently, and the infected cells can initiate tumorigenesis, including estrogen receptor-positive and hormone-dependent tumors, which are the most common subtype of human breast cancer but are yet still difficult to model in mice. This technique provides another tool for studying formation, prevention, and treatment of breast cancer, especially estrogen receptor-positive breast cancer.

Introduction

There are many mouse models of breast cancer, including conventional xenograft models, carcinogen-induced tumors, intraductal injection of breast tumor cells, genetically engineered mice, and genetic alterations of mammary epithelial cells by intraductal injection of viral vectors [1–8]. These models have greatly advanced our understanding of breast cancer formation, progression, and therapy and therapeutic resistance. However, a mouse mammary gland is quite dissimilar from a human breast: the mammary ductal tree in a sexually mature non-lactating mouse terminates in blunt ductules, and lobuloalveolar development towards terminal ductal lobular units (TDLUs) occurs only during pregnancy and largely regresses following involution. In contrast, the mammary ductal tree in a sexually mature non-lactating woman already ends in TDLUs that harbor extensive amounts of acini [9]. Furthermore, the mouse mammary stroma is largely comprised of adipose tissue with scanty connective tissue, while the human breast stroma contains dense amounts of collagenous connective tissue beside the adipose tissue [9, 10].

Compared to mammary glands of mice, those of rats are more similar to the human breast – in sexually mature rats, a ductal tree terminates in TDLUs and shows extensive alveolar development even in the absence of pregnancy [9], and the mammary stroma also contains, besides the adipose component, a significant proportion of connective tissue and fibroblasts organized as a sheath around TDLUs [11]. Therefore, rats are potentially better than mice for modeling human breast cancer. Another important advantage of rat models is that rats develop estrogen receptor-positive (ER+) cancer readily [12–15]. ER+ breast cancer accounts for approximately 70% of all human breast cancer cases, but among the large number of mouse models, only a few (excluding xenograft models) develop ER+ cancer, with uncertain response to hormone therapy [16–19, 6, 20–23]. In contrast, intragastric injection of a carcinogen such as 7,12-dimethylbenz(a)anthracene (DMBA) or N-methyl-N-nitrosourea (NMU) into rats readily leads to ER+ and hormone-dependent mammary tumors [12–15]. A significant proportion of the resulting tumors harbor mutations in Ras genes [24]. Nevertheless, the uncertainty of the initiating genetic mutations has diminished the appeal of these carcinogen models for studying cancer initiation, progression, and treatment. Genetic engineering techniques can introduce precise genetic alterations into the genome and can overcome the genetic uncertainty of carcinogen models. For example, germline deletion of NF1 in rats leads to the formation of ER+ mammary tumors [25]. However, germline manipulation of proto-oncogenes and tumor suppressor genes often affects normal mammary gland development [26]. On the other hand, intraductal injection of virus can deliver oncogenes into a small number of mammary cells in a fully developed normal mammary gland to initiate tumorigenesis [27, 28, 5]. Intraductal injection of retrovirus carrying either Ras or ErbB2 has been reported to induce ER+ mammary tumors in rats, but these reports used antipsychotic drugs such as perphenazine to increase serum prolactin, to force mammary cell proliferation, and to promote retroviral infection rates, complicating model generation and data interpretation [29–31].

Here, we describe in detail the use of lentivirus to infect fully developed mammary glands of rats. This method infects mammary epithelial cells efficiently, and oncogenes such as mutated Ras or mutated PIK3CA, introduced by a lentiviral vector, can lead to mammary tumors. Of note, a mutated Ras gene introduced by this method leads to ER+ and hormone-dependent mammary tumors, and a mutated PIK3CA also leads to ER+ tumors (hormone dependency not yet tested). Therefore, this technique provides a valuable tool for modeling formation, prevention, and treatment of human breast cancer, especially ER+ breast cancer.

Materials

Plasmids:

The FUCGW lentiviral vector [32] is used as a vector to carry an oncogene, GFP, luciferase, or other genes of interest. This “gutless” vector needs to be co-transfected with plasmids encoding the viral accessory components (pREV, pMDL, pVSVG) to make infectious but replication incompetent lentivirus. FUCGW carrying Hras mutated at codon 61, FUCGW-HrasQ61L, has been previously reported [33].

Cells:

293T cells (ATCC, CRL-3216) are cultured in DMEM medium (Corning, 10-013-CV) supplemented with 10% FBS and penicillin/streptomycin.

Transfection reagents:

jetPRIME (Polyplus-transfection, ref. # 114-01) is used for transfecting lentiviral vectors into 293T to make lentivirus.

Animals:

Any rats older than 5 weeks of age may be used for intraductal injection of lentivirus. Rats older than 6 months may have collapsed ducts that are difficult for virus to permeabilize. White rats are easier for monitoring the success of injection than black rats. Examples of rats we used include white Sprague Dawley and agouti Long-Evans rats (Charles River, Strain Code: 400 and 006, respectively).

Drugs/chemicals:

Bromophenol blue (Fisher, BP114-25) in trace amounts is added to the viral solution to be injected, so as to visualize the success of the intraductal injection. Artificial Tears 15/83% Ophthalmic Ointment Sterile 3.5gm/Tb (Henry Schein, 1371627) is used to protect the eyes while the rat is under anesthesia.

Tools:

One 0.45 µm PVDF filter (Millipore # SCHV01RE); one pair of Vannas Micro Dissecting Spring Scissors (Roboz, RS-5621); one 33-gauge metal hub needle (Hamilton, Part # 90033; while keeping the manufacturer-provided fine metal rod inside the needle, use a pair of sharp scissors to cut the needle to 1 cm in length); one 50 µl gastight syringe (Hamilton, Model 1705 TLL, PTFE Luer Lock, Part # 80920); and one fluorescent stereomicroscope (e. g., Leica, model MZ16 F).

Antibodies:

Rabbit anti-GFP antibody (Cell Signaling, cat # 2956s) is used to evaluate success of infection. The rabbit anti-ERα antibody (MC-20; Santa Cruz, cat # sc-542) is used to detect ER status of rat tumors. The rabbit anti-PR antibody (Dako, cat # A0098) is used to determine the PR status of rat tumors.

Methods

Lentivirus preparation:

Lentivirus is prepared by transfecting the FUCGW plasmid [32] carrying the gene of interest along with plasmids encoding the viral accessory components into 293T cells using calcium phosphate as previously described [5], or using the jetPRIME transfection reagent as follows:

  1. Dilute the FUCGW lentiviral construct and the three accessory components (pREV, pMDLg, pVSVG) to concentrations of 1,000 ng/µl.

  2. Day 1: Plate 293T cells at 1.2 x 107 cells per 15 cm dish in 20 ml DMEM supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. Incubate the dishes overnight at 37°C.

  3. Day 2: When the confluency is at 60–80%, they are ready for transfection. The following transfection mixture is for twelve 15 cm dishes and can be scaled up or down at needed.

  4. Add 12 ml jetPRIME buffer to a 50 ml conical tube.

  5. Add 120 µl of FUCGW and 40 µl each of pREV, pMDL, and pVSVG. Mix by vortexing for 5 seconds.

  6. Vortex the jetPRIME solution for 5 seconds and spin it down. Add 480 µl to the DNA mixture tube above. Vortex this transfection mixture for 10 seconds and spin it down.

  7. Incubate this transfection mixture for 10 minutes at room temperature.

  8. Transfer two 293T culture dishes from the incubator to the tissue culture hood. Leave all the medium in. Use a p1000 pipetman to add 1,020 µl of the transfection mixture slowly and dropwise to each dish while gently rocking the dish. Return these dishes to the incubator.

  9. Repeat this for the remaining dishes two at a time.

  10. Allow the dishes to incubate for 4 hours so that the transfection can complete.

  11. Replace the medium with pre-warmed fresh culture medium (20 ml per dish).

  12. Day 4: Check GFP expression under a fluorescent microscope. Should see green fluorescence in >80% cells. Harvest the virus by collecting culture supernatant. Add 20 ml of prewarmed new medium and return the dishes to the incubator. Store the collected media at 4 °C.

  13. Day 5: Harvest culture supernatant again. Filter the two batches of supernatant through 0.45 µm PVDF membranes.

Virus concentration and storage:

Virus is unstable at 4°C and should be concentrated and frozen as soon as the collection is complete. The virus in the medium is spun down by ultracentrifugation (27,000 rpm in the SW32 rotor at 4 °C for 90 minutes). Gently vacuum suck the supernatant without disturbing the pellet. Leave 1/100th to 1/200th of the supernatant in the centrifuge tube (for 35 ml of culture supernatant, leave 175 µl to 350 µl). Pipette rigorously for approximately 2 minutes to resuspend the virus, but do not vortex as vortexing may lead to a viral titer loss. Combine the virus from all centrifuge tubes into one tube. Swirl the tube 10 times to mix solution. Aliquot into 50 µl per 0.5 ml microfuge tube. Freeze at −80 °C. Prior to the intraductal injection, the viral tubes should be transferred to, and kept on, ice.

Virus titer determination:

Take one frozen viral tube and determine the viral titer as previously described [5]. As viral titer drops slightly upon a cycle of freeze-and-thaw, it is important to use a frozen tube rather than the freshly concentrated virus for titer determination, so that the titer most accurately reflects the titer of the virus that will be used for intraductal injection. A titer of 108 – 109 infectious units per ml is viewed to be adequate for intraductal injection.

Lentiviruses produced by this method can infect humans, and the oncogenes engineered by these lentiviral vectors introduce additional risk to laboratory personnel. Therefore, transfection, virus collection, and aliquoting should all be handled cautiously in a biosafety level 2 (BSL-2) tissue culture hood. Gloves should be worn, and other personal protective equipment should be considered.

Intraductal injection of lentivirus into rat mammary glands:

The up-the-teat injection of lentivirus into rat mammary glands is very similar to the method used in mice, which has been previously described in detail [34, 27], but there are some notable differences. First, unlike mice, which have 5 pairs of mammary glands, rats have 6 pairs of mammary glands. Any of these teats can be used for intraductal injection of virus. We usually use the #2~5 glands in rats for convenience. Another difference between mice and rats is that the mammary ductal tree of rats is much larger than that of mice, so the volume of the virus injected into each rat mammary gland can be larger than the maximum volume of approximately 10–30 µl in mice depending upon the strain and age. 60 µl of virus can be injected intraductally with ease into rats. Furthermore, since the milk duct opening of the rat teat is much larger than that of the mouse, a magnifier or a dissecting scope is generally not needed to aid the injection, unlike intraductal injection of mice.

Below is a brief description of the rat intraductal injection method. All rat experiments should be performed under an institutional animal care and use committee-approved animal protocol.

  1. Thaw desired numbers of frozen tubes of lentiviral stocks depending on the number of rats to be injected. Combine the contents into a single tube to minimize variations. Use the tip of a syringe needle to pick up a trace amount of bromophenol blue powder, add it to the virus solution, and mix well, so that the injected virus can be visualized through the unexposed skin (easier with white rats than black rats). Keep the virus on ice at all times.

  2. Anesthetize rats and inject virus one at a time. A precision vaporizer that delivers isoflurane at 2–3% is preferred as the rats wake up as soon as isoflurane stops flowing to the nose cone. Alternatively, an anesthesia combination solution that contains 42.8 mg ketamine, 8.6 mg xylazine, and 1.4 mg acepromazine per ml may be used. This solution is injected intraperitoneally at 0.75 −1.5 ml/kg body weight. Apply a small amount of Artificial Tears to protect the eyes while the rat is under anesthesia.

  3. Place an anesthetized rat in the supine position, and spread out the fore- and hind-limb using adhesive tape. Use 70% alcohol to move the hair away from the teat to be injected.

  4. Transect the most distal tip of the teat to expose the opening of the lactiferous duct using a pair of Vannas Micro Dissecting Spring Scissors.

  5. Draw up to 30 μl of bromophenol blue dyed-virus into a 50 µl gastight syringe fitted with a 33-gauge blunt needle.

  6. Gently insert the needle 1–2 mm into the lactiferous duct in the teat while taking caution not to pierce the duct (Figure 1A). Then, slowly inject the viral solution into the duct and observe the dye permeabilizing the ductal tree (Supplemental video). The dye spread in rats is much more difficult to observe than in mice as the rat skin is much thicker.

  7. Additional teats of the rat may be injected as needed.

  8. Keep the injected rat on a warm pad until it regains consciousness fully.

  9. Repeat the above the procedure for each additional rat.

  10. Discard virus tubes and other solid waste in contact with the virus in a biohazard waste receptacle. The syringe and needle should be thoroughly cleansed with 70% alcohol.

Figure 1.

Figure 1.

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Intraductal injection of lentivirus to infect rat mammary epithelial cells. (A) Method of intraductal injection. The picture shows the injection of virus into the ductal opening of the teat. (B) Photomicrography of the green fluorescent signal of a whole mammary gland under a fluorescent stereomicroscope. Both an un-injected gland (the right picture) and a gland injected with lentivirus carrying EGFP gene three days earlier (the left picture) are shown. (C) Immunohistochemical staining for EGFP to detect the infected cells. An un-injected gland was used as a negative control. (D and E) Detection of infected cells by flow cytometry. The FUCGW-infected #4 mammary gland was digested into single cell suspension and analyzed by flow cytometry to detect EGFP+ cells three days after intraductal injection. Representative flow cytometry analyses of cells from both injected and uninjected glands are shown in D. The estimated percentage of infected cells based on flow cytometry analysis of EGFP+ cells is shown in E. Long-Evans rats, age = 8 weeks, n = 3. Virus dose: 1.4x108 IUs/gland.

The FUCGW virus, which carries the gene encoding the fluorescent reporter EGFP, may be injected into separate teats or separate rats along with the test virus to determine the infection efficiency or serve as a control. The EGFP signal can be readily detected 2–3 days after intraductal injection under a fluorescence stereomicroscope. An example is shown in Figure 1B. Immunohistochemistry for EGFP may also be performed to locate the infected cells. An example of GFP detection in both ducts and alveoli is shown in Figure 1C. In addition, flow cytometry may be used to quantify infection rates (Figure 1D). Injection of 1.4x108 IUs/gland leads to an infection rate of approximately 0.4% (Figure 1E).

Generation of ER+ mammary tumors through intraductal injection of lentivirus carrying mutated Hras:

To test whether ER+ mammary tumors can be efficiently generated through intraductal delivery of lentivirus carrying an oncogene, we used intraductal injection of the lentiviral vector FUCGW carrying the mutated oncogene HrasQ61L to infect mammary epithelial cells of Spraque Dawley rats at the age of eight weeks (7x107 IUs/gland). This oncogene was selected due to the known role of v-Hras in inducing ER+ tumors in rats [29], although this particular Q61L mutation has not been tested directly in rats. As shown in Figure 2A, tumors appeared with a median time of 25 days, a few weeks faster than tumorigenesis initiated by retroviruses carrying v-Hras [31, 30]. The tumors induced by FUCGW-HrasQ61L are adenocarcinomas (Figure 2B). Immunohistochemistry for ER and progesterone receptor (PR) shows that these tumors are highly positive for both ER and PR (Figure 2C).

Figure 2.

Figure 2.

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Hormone receptor-positive and hormone-dependent mammary tumors induced by intraductal injection of lentivirus carrying HrasQ61L. (A) Kaplan-Meier plot of tumor latency of rats intraductally injected with FUCGW-HrasQ61L. Sprague Dawley rats (n=26) at the age of 10 weeks were injected intraductally with 7x107 IUs of FUCGW-HrasQ61L into one gland per rat. Contralateral un-injected glands were used as negative controls. (B) Representative H&E staining of tumors induced by FUCGW-HrasQ61L. (C) Representative ER (left) and PR (right) immunohistochemical staining of tumors induced by FUCGW-HrasQ61L. (D) Tumor volume changes at 2 weeks after ovariectomy or sham surgery. (E) Tumor volume changes over extended periods of time. Hormone-dependency is lost over time in a subset of rats. The black lines are tumors from sham surgery control rats; the red lines are tumors from ovariectomized rats.

Testing of hormone dependence of mutated Hras-induced tumors:

To determine whether these HrasQ61L-induced tumors are hormone-dependent, we performed ovariectomy or sham surgery (10 each) on rats carrying a tumor. As shown in Figure 2D, while tumors in the sham group did not shrink after the surgery, tumors in the ovariectomized rats collapsed with the first two weeks. However, some of the residual tumors resumed growth over time (Figure 2E), similar to ER+ breast cancer in patients undergoing anti-hormone therapy. Therefore, this rat model of breast cancer may be useful for investigating mechanisms of hormone therapy and preclinical testing of new hormone therapeutics. Of note, some of the tumors in ovariectomized rats started to grow within one month, much faster than the typical speed of therapeutic resistance observed in women under anti-hormone therapy. While the exact reason is not clear, one possibility is that these tumors are likely multi-clonal so that one or more clones could be ovarian hormone-independent and expand quickly following ovariectomy.

Discussion

Here we describe an efficient method for delivering an oncogene via a lentiviral vector into the rat mammary epithelia for modeling of human breast cancer. This method allows genetic manipulations of small numbers of mammary cells in fully developed mammary glands so as to closely mimic sporadic breast cancer development in women. Tumors arise only in injected mammary glands, so that the non-injected glands may be used as experimental baseline controls. We have found that intraductal injection of either HRasG61L or PIK3CAH1047R leads to mammary tumors with median latency of 4 weeks or a few months, respectively. These tumors are ER+/PR+, providing much needed models of human ER+ breast cancer that are rare in mouse models of human breast cancer. We are currently building additional rat models using other oncogenes as drivers of tumorigenesis.

This intraductal viral injection method may also be used to deliver Cre to excise tumor suppressors and other genes that are flanked by loxP sites. Likewise, it may be used to introduce shRNA to suppress the expression of selected genes in mammary epithelial cells. In addition, this method may be adopted to introduce gRNA to edit endogenous genes.

This intraductal viral injection approach may be used to infect the mammary glands at selected developmental times (pubertal, mature, various stages of pregnancy, lactation, involution, or post-involution) to investigate how mammary gland development and reproduction may affect breast cancer risk and progression. We have previously used this method in mice to study the impact of pregnancy [35, 36] and certain hyperprolactinemia-inducing drugs [37] on breast cancer risk. Besides, this approach may be used to study genes affecting mammary gland development in rats, as we have reported in mice [38, 39].

Commonly used lentiviruses including FUCGW infect any cells that they are in direct contact with. However, since the virus is injected into the ductal lumen which is sealed off by basement membrane from the stromal component, the injected virus infects predominantly the luminal epithelial cells and possibly a few basal cells, but not stromal cells. The virus is not expected to leak into the bloodstream to infect cells in other tissues.

We have previously reported techniques to infect selected mammary epithelial cells in mice [3]. We engineered mice that express the gene encoding the avian leukosis virus (ALV) receptor TVA in specific subsets of mammary cells, such as certain progenitor cells [40] or differentiated epithelial cells [35], and used intraductal injection of an ALV-derived vector (RCAS) to infect TVA+ cells selectively to achieve cell type-specific expression of genes of interest for studying mammary development and tumorigenesis [35, 41, 33, 37, 42–47, 36, 48, 49, 40, 50, 51, 38]. We have also used the ALV env gene-pseudotyped lentivirus to infect these tva transgenic mice to achieve cell type-specific expression [4]. Therefore, if selected cells need to be infected in rats, tva transgenic rats may be generated and utilized together with RCAS or env-pseudotyped lentiviral vectors to achieve infection with spatial controls.

In summary, the intraductal lentiviral infection method described here provides a very efficient tool to deliver genes into mammary epithelial cells in rats at a selected time. Mammary tumors, including ER+ and hormone-dependent tumors, can be induced readily and quickly by delivering an oncogene through this method. Therefore, this technique provides a valuable tool for modeling formation, prevention, and treatment of human breast cancer, especially ER+ breast cancer.

Supplementary Material

1645321_Vid1

Supplemental video

This video shows the intraductal injection process. The right #4 gland of a Long-Evans rat at the age of 12 weeks was being injected.

Download video file (91.2MB, mp4)

Acknowledgments

This work was supported in part by National Institutes of Health grants R01CA204926 (Y.L.), R01CA205594 (Y.L.), and P50CA186784 (Y.L.), and Department of Defense grants BC160240P1 and BC191649 (Y.L.).

Footnotes

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

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Supplementary Materials

1645321_Vid1

Supplemental video

This video shows the intraductal injection process. The right #4 gland of a Long-Evans rat at the age of 12 weeks was being injected.

Download video file (91.2MB, mp4)

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