From the Department of Biophysics, Biological Faculty, Moscow
State University, 119889, the § Biophysical Laboratory and the
Laboratory of Genetic Engineering and Molecular Diagnostics of
Microorganisms, Russian Institute of Agricultural Biotechnology,
127550, Timiryazevskaya Street, 42, and the ** Belozersky Institute of
Physico-Chemical Biology, Moscow State University, 119899, Moscow,
Russia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transfection of HC-11 murine epithelial mammary
cells as well as murine and sheep mammary glands were carried out using
insulin-containing constructs that deliver DNA by receptor-mediated
endocytosis to receptor-expressing cells. In vivo
transfection of mammary gland tissue with the luciferase gene was
carried out by introducing the DNA constructs into the mammary ducts of
both mice and sheep. The successful transfection of ewe mammary glands
was demonstrated by the detection of luciferase activity in mammary
gland biopsy material up to a month after a single administration of
the construct. To test whether products of expression of transfected
genes could be secreted into the milk in this system, the N-terminal
secretory signal sequences of bovine -lactoglobulin or the entire
coding sequence of human
-lactalbumin were fused to the N terminus
of the luciferase gene. After transfection with the modified
luciferases, both murine and sheep milk could be shown to contain
luciferase activity, whereas mice, which had been transfected with the
nonmodified luciferase gene, did not secrete any activity in the milk.
This approach demonstrates for the first time the possibility of gene transfer in vivo into mammary gland epithelial cells using
constructs delivering DNA via receptor-mediated endocytosis.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three major approaches are currently being used to achieve gene delivery into recipient organs: (i) animal virus-based vectors (retroviruses and adenoviruses), (ii) liposomal transfer, and (iii) receptor-mediated endocytosis (for review, see Refs. 1-6). In terms of wide application in human gene therapy, the first approach is limited by safety concerns with respect to the use of viral vectors, whereas the second approach has disadvantages associated with the cytotoxicity of Lipofectin for some organs and in vivo instability of liposomes. Since gene delivery via receptor-mediated endocytosis does not make use either of viral vectors or liposomes, it appears to be the method of choice for gene transfer in vivo, although its application in human gene therapy thus far has been largely restricted by problems of high variability and lower efficiency of gene transfer, compared with the first two methods.
These problems associated with receptor-mediated gene delivery are attributable to the fact that most parameters determining the efficiency of this process have not been characterized or optimized. In particular, the methods of preparation of DNA-containing complexes, the specificity of the receptor selected for delivery, the specific properties of target tissue, etc. may all be critical. To date very few receptors have been used for DNA delivery via receptor-mediated endocytosis, including asialoglycoprotein (7-10), transferrin (11), polymeric immunoglobulin (12), and several other (13, 14) receptors, whereas in vivo studies have been restricted to only two organs, liver and lung (8, 12, 15-24). A serious problem with respect to many methods of gene delivery is the degradation of DNA in cellular lysosomes before it reaches the nuclei of transfected cells. Wu and co-workers (16, 17) showed that the long term persistence and expression of a foreign gene in rat liver can occur when partial hepatectomy is performed after gene delivery, which is suggested to be through partial hepatectomy, disrupting the hepatocyte microtubular network which in turn blocks the progression of DNA-containing endosomes to lysosomes for degradation (24).
To rescue DNA from cellular lysosomes, some authors have suggested the use of DNA-delivering constructs coupled with mutant adenoviruses (25, 26) or the addition of amphipathic peptides (27) or a lysosomotropic agent such as chloroquine (28), although the latter may have only limited application because of the known toxicity of the inhibitor. Although impressive data on direct gene transfer to airway lung epithelium employing adenovirus·polylysine·DNA complexes have been obtained (21), it is not yet clear how far this approach may be extended to other systems of gene delivery via receptor-mediated gene uptake. Thus, the scarcity of reports appearing to date on in vivo receptor-mediated transfection makes their generalization impossible.
We have previously investigated the use of insulin and the insulin receptor for gene delivery and demonstrated in vitro gene transfer by an (Ins-pLys)-DNA1 construct via the receptors of cultured cells (29-32).2 The insulin receptor is known to be widely represented in almost all tissues of mammals, which makes it promising for local gene transfection to virtually any organ. In the present report, we demonstrate for the first time in vivo insulin receptor-mediated gene transfer to mammary glands of mice and ewes. We analyze the effect of co-transfection of the mammary gland with avian and attenuated human adenoviruses on the level of gene expression (luciferase) and its persistence for several weeks. In addition, we demonstrate the feasibility of using receptor-mediated transfection of mammary gland tissue to enable the products of foreign genes to be secreted in the milk of transgenic animals. Our results have relevance both to gene therapy approaches and the potential production of proteins of importance in the milk of large mammals such as dairy cattle.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Cultures
Murine mammary epithelial HC-11 cells were grown (33) at 37 °C in RPMI 1640 medium with 10% fetal calf serum and 50 µg/ml gentamycin using a humidified atmosphere with 5% CO2 and supplemented with 5 µg/ml insulin and 10 ng/ml epidermal growth factor (all Sigma).
Animals
18-20 g of female BALB/c mice and 3-year-old Romanov ewes were used.
Synthesis and Purification of Conjugates
Insulin-Polylysine-- Bovine insulin (Sigma) was 125I-iodinated with the use of IODO-GEN (Pierce). 90-kDa poly-L-lysine (pLys; Sigma) was labeled with 1-fluoro-2,4-dinitro-[U-14C]benzene (Amersham Pharmacia Biotech). The preparation and purification of the conjugate were carried out as described previously (29); briefly, bovine [125I]iodoinsulin conjugated with citraconic anhydride (Serva) to protect its terminal amino groups was covalently linked to [14C]pLys with the use of the bifunctional cross-linking reagent, N-succinimidyl-3-(2-pyridyldithio) propionate (Sigma) according to Jung et al. (34). The resultant conjugate was purified on a Sephacryl S-200 (Amersham) column, and the citraconyl groups were then removed. The insulin/pLys molar ratio in different batches of the conjugate was between 5 and 10 insulin molecules per molecule of pLys. About 80% of the insulin reacted with the pLys, with the yield after purification procedure being ~50%.
Streptavidin-Polylysine (Str-pLys)-- Streptavidin (Molecular Probes) was linked to 90-kDa pLys with the use of N-succinimidyl-3-(2-pyridyldithio) propionate using the same procedure as that described above without the terminal amino group protection, and the conjugate was purified on a Sephacryl S-300 column. The pLys/streptavidin molar ratio was between 1 and 2 pLys molecules per molecule of streptavidin.
Preparation of Secretory Forms of Luciferase Gene
The initial plasmid was pRSVL, kindly provided by Dr. D. Helinski (35). In this vector, the luciferase gene is under control of the long terminal repeat of Rous sarcoma virus. To prepare plasmid pRSV-spBLG-luc and all other constructions, site-directed mutagenesis was used (36). Briefly, plasmids were digested with a restriction endonuclease, cutting at a unique site in the vicinity of the site selected for mutation and then treated with T4 DNA polymerase in the absence of dNTP to digest one strand of each end of the cleaved DNA. A mutagenic primer and two adaptor primers were then annealed to the single-stranded tails thus formed. The adaptors were sequences complementary to the opposite strands of DNA, which were needed to restore the sticky ends at the initial site of plasmid cleavage. The adaptor complementary to the strand to be mutagenized remained unphosphorylated at the 5'-end. The subsequent addition of T4 DNA ligase and T4 DNA polymerase in the presence of dNTP restored the initial site of plasmid cleavage, filled the gaps, and closed the mutant strand into a circle. The wild type strand remained open because of the nonphosphorylated adaptor. Finally, the second mutant strand was formed by nick translation using Escherichia coli DNA polymerase. After transformation, the yield of mutants was normally about 100%.
To construct the plasmid with a signal peptide, the HindIII site of pRSVL was used with the adaptors 5'-GTGTGCACCTCCA-3' and 5'-GTACCGGAATGCTA-3' for the upper and bottom DNA strands, respectively. The following mutagenic oligonucleotide was used to prepare pRSV-spBLG-luc: 5'-CATCCTCTAG AGGATAGAAT GGCGCCGGGC CTTTCTTTAT GTTTTTGGCG TCTTCGACGA TGATGGCC-3'.
To construct the plasmids of the pCMV series, HindIII-SmaI fragments of plasmid pRSVL (comprising the whole luciferase gene together with its 5'- and 3'-untranslated regions) were subcloned into the HindIII and EcoRV sites of plasmid pCRTM3 (Invitrogen). This vector permits expression of a protein from the strong cytomegalovirus (CMV) promoter. This generated the pCMV-luc plasmid. To generate the construct encoding the LA luciferase fusion protein, an NheI restriction site was introduced immediately upstream of the luciferase gene in pCMV-luc using the mutagenic primer 5'-GTTTTTGGCGT CTTCCGCTAG CATGGTTTAC CAACA-3' by the same method of mutagenesis (36), resulting in an NheI site containing pCMV-luc vector. In parallel, the human LA gene was synthesized by polymerase chain reaction using human genomic DNA as a template. The primers for polymerase chain reaction contained BglII and NheI restriction sites for subsequent subcloning of the polymerase chain reaction product. The human LA gene thus obtained was then cloned into pGEM7Zf(+) vector (Promega), and the sequence was confirmed. Finally, the LA gene was excised using HindIII and NheI restriction sites and inserted into the modified NheI site containing pCMV-luc vector to generate plasmid pCMV-LA-luc.
Adenoviruses
Duck egg drop syndrome (EDS-76) virus was grown as described (37). Chicken embryo lethal orphan virus was grown in 9-day-old chicken embryos; after a 3-day incubation, virus-containing allantois fluid was harvested (37). Human adenovirus type 5 (Ad5) dl312 strain was grown in the 293 cell line. After incubation, infected cells were harvested, centrifuged, homogenized, mixed with an equal volume of Freon 113, and then recentrifuged. The aqueous phase was used for virus purification.
Virus-containing material was centrifuged through a discontinuous CsCl gradient. The viral bands were collected and centrifuged through a preformed linear gradient of CsCl. The viral bands were again collected and stored at 4 °C. Virion concentration was determined by spectrophotometric analysis (38).
Virions (5 × 1011 virions/ml in 1.36 g/ml CsCl solution containing 150 mM NaCl, 25 mM HEPES, pH 7.8) were biotinylated (3 h at room temperature) with 7 µM biotinamidocaproate N-hydroxysuccinamide ester (Sigma) followed by dialysis against 25 mM HEPES, pH 7.5, containing 0.15 M NaCl.
Preparation of Ins-pLys Plasmid DNA-transfecting Construct
Complex formation of Ins-pLys conjugate with plasmid DNA was carried out as described previously (31). Briefly, Ins-pLys conjugate was added dropwise to the plasmid solution in 0.25 mM EDTA, pH 8.0, with mixing. Formation of virus-containing complexes was accomplished at pH 7.5 in 25 mM HEPES buffer with 150 mM NaCl and 0.25 mM EDTA by the sequential addition of Str-pLys, plasmid, and Ins-pLys to biotinylated viruses.
Binding and Internalization of the Transfecting Construct
Internalization of fluorescein isothiocyanate-labeled (31) Ins-pLys plasmid construct was assessed by video-intensified microscopy using an AT200 cooled CCD camera (Photometrics) and Axioplan microscope (Zeiss).
Transfection of Cultured Cells
HC-11 cells were plated 2 days before an experiment. After washing with fresh RPMI 1640 medium, transfecting complexes (1-12.5 nM DNA) in RPMI 1640 medium with 2 mg/ml bovine serum albumin buffered with HEPES, pH 7.5, were added to the cells and incubated for 3 or 18 h at 37 °C. A 5-fold excess of RPMI 1640 medium supplemented with 5% fetal calf serum and bicarbonate was then added, and the cells were placed in a CO2 incubator for 2 days unless otherwise indicated.
Transfection of Mammary Glands
A special device to introduce material into the milk duct of a mammary gland was constructed (32).2 Briefly, the device consisted of a body with a nipple-shaped hollow, a channel for delivery of negative pressure, a special sphincter dilator with an axial channel in the bottom of the hollow; it also had a special cannula, with a cavity to measure volumes that passed through the axial channel (32).2 25-75 µl and 75 ml of transfecting complexes in Hanks' solution containing 50 µg/ml gentamycin were introduced into the milk ducts per murine mammary gland and sheep mammary gland, respectively. Lactation of ewes was induced according to a 2-week regime of hormonal induction (39).
Luciferase Assay
Luciferase activity in cultured cells was assayed in the presence of CoA (40). Cells were washed with 100 mM potassium phosphate buffer, pH 7.8, containing 1 mM dithiothreitol and 2 mM EDTA, disrupted using 1% Triton X-100 (Serva) in the same buffer, and centrifuged. 50 µl of the resultant supernatant were mixed with 350 µl of 25 mM glycylglycine buffer, pH 7.8, containing 15 mM MgSO4, 2 mM ATP, 10 mM dithiothreitol, then 20 µl of 1 mM CoA and 100 µl of 1 mM luciferin were added, and the luminescence was measured using an LKB 1250 luminometer. A standard of 1 ng of luciferase (Sigma) corresponds to 1250 AU.
Mammary glands were washed with Hanks' solution, homogenized in 100 mM potassium phosphate buffer, pH 7.8, with 1 mM EGTA, 3 mM Mg(CH3COO)2, 0.1% bovine serum albumin, 1 mM dithiothreitol, 1% Triton X-100, 20 µg/ml aprotinin (Sigma), and centrifuged. The luciferase activity was measured in the obtained supernatant as above. Biopsy material was frozen and kept in a liquid nitrogen until use.
Western Blot (Immunoblot) Analysis
Milk proteins (10 µl of milk/lane) were resolved by sodium dodecyl sulfate polyacrylamide gel (12.5%) electrophoresis and transferred to nitrocellulose membranes. Membranes were incubated at 25 °C for 60 min in Tris-buffered saline containing 0.05% Tween 20 and 3% bovine serum albumin and then for 60 min with a primary antibody. We used affinity-purified rabbit anti-luc obtained from Promega diluted 1:5000. After being washed with Tris-buffered Tween saline, membranes were incubated for 60 min with anti-rabbit IgG alkaline phosphatase conjugate (Sigma), and the immunoblots were finally developed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma) as substrate.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Internalization of Ins-pLys Plasmid DNA Constructs-- We had previously used insulin-containing conjugates to target foreign genetic materials (29-32)2 and photosensitizers (41-43) into hepatoma cells and, using confocal laser scanning microscopy and video-intensified microscopy, had been able to directly visualize that the constructs could be endocytosed. Murine mammary epithelial HC-11 cells possess insulin receptors (data not shown) and are thus able to bind and internalize insulin molecules. The fluorescein isothiocyanate-labeled Ins-pLys plasmid construct was incubated with HC-11 cells for 3 h, and free excess insulin inhibited binding and internalization of the construct (Fig. 1a-d). Video-intensified microscopy of a single cell with higher magnification reveals that the construct was localized in the cytoplasm near the cell nuclei (Fig. 1e). The image was processed using a gradient Sobel image filter, which resulted in the direction-independent enhancement of brightness gradients in the image (44). This processing revealed that the construct appeared to be localized in vesicular and/or tubular bodies, probably endocytotic compartments (Fig. 1f).
|
Transfection of HC-11 Cells in the Absence and Presence of Adenoviruses-- The (Ins-pLys)-pRSVL construct (12.5 nM plasmid DNA and a lysine/nucleotide ratio of 0.4) was used to transfect HC-11 cells, with luciferase activity readily detectable in cell lysates (4.9 ± 0.7 AU/106 cells). Excess free insulin almost completely inhibited the transfection (less than 0.5 AU).
Endocytosed DNA is known to face many obstacles before it reaches the nucleus, one of which is degradation in endosomes/lysosomes. Adenoviruses are able to exit from endosomes along with other components of these vesicles, and this property of adenoviruses has been employed successfully to enhance the transformation efficiency of the transfecting constructs (25-26). We decided to test whether the replication-deficient human adenovirus, serotype 5, strain dl312 (Ad5) adenoviruses could increase transfection of mammary gland epithelial cells using our insulin-containing constructs. A significant increase in cellular luciferase activity was obtained when the virus was coupled directly to pLys through a streptavidin-biotin linkage; the activity was dependent on the Str-pLys/virion ratio in the construct (Fig. 2A). Free Ad5 was less efficient when co-incubated with (Ins-pLys)-pRSVL construct; for example, the addition of 3.3 × 1010 virions/6-cm dish 60-fold increased transfection of HC-11 cells by 5.1 nM Ins-pLys construct. Greater addition of Ad5 led to cytotoxicity. Excess free insulin added together with the transfecting construct significantly decreased reporter gene expression, again indicating that transfection in this system was mainly an insulin-receptor-mediated process (Fig. 2B). Avian adenoviruses (chicken embryo lethal orphan virus or EDS-76) lacking determinants for mammalian cells were also tested and found to be able to enhance HC-11 cell transfection provided they were linked to the transfecting constructs. Free avian viruses, in contrast to human Ad5, were unable to enhance transfection (data not shown).
|
In Vivo Transfection of Mammary Glands-- The (Ins-pLys)-pRSVL construct was infused into the milk ducts of mice. On days 1, 3, 6, and 9 the mice were sacrificed, their transfected and intact mammary glands and livers were homogenized, and luciferase activity was measured. Luciferase activity could be detected in extracts prepared from the transfected mammary glands over all 9 days, whereas no activity could be detected in the intact glands (Fig. 3).
|
|
|
|
Constructs for Secreted Forms of Firefly Luciferase--
Though
luciferase is a very commonly used reporter gene/protein due to the
high sensitivity and simplicity of assay of its enzymatic activity, it
is not such an attractive reporter in the case of the mammary gland,
since firefly luciferase is not secreted from transfected cells. To
obtain secreted forms of firefly luciferase, several approaches were
taken. The signal sequence from ovine -lactoglobulin was added to
the N terminus (MEDAKN6) of luciferase between the first
and second amino acids (protein encoded by plasmid pRSV-spBLG-luc). The
luciferase gene was also fused at its N terminus with the whole gene of
human LA (see "Experimental Procedures").
|
In Vivo Transfection of Mammary Glands with Modified Luciferase Genes-- In vivo transfection of mouse mammary glands with (biotinylated EDS-Str-pLys)-(pRSV-spBLG-luc plasmid)-(Ins-pLys) (2 nM) by infusion 3-4 times into milk ducts of pregnant mice (second half of pregnancy) at 1-day intervals resulted in significant luciferase activity (mean of 118 ± 13 AU/ml, n = 6) secreted in milk. There was no luciferase activity in the milk of mice transfected with the same procedure with pRSVL plasmid.
For sheep mammary gland transfection, pCMV-LA-luc (30 nM) was used. Three ewes were injected with hormones for 14 days; 75 ml of the transfecting construct were introduced into one mammary gland of each on day 10 and into both mammary glands of each on day 12; meaning that every animal had one gland transfected once and one gland transfected twice. Two of the three sheep began to lactate, producing more than 200 ml/milking and enabling secreted luciferase activity to be quantified. Mammary glands transfected once produced detectable luciferase activity only on day 5 from the beginning of lactation, and this activity was very low, ~2 AU/ml (data not shown). The luciferase activity in the milk of mammary glands transfected twice was significantly higher, peaking on days 4 and 5 (Fig. 7). Immunoblotting indicated the expected 75-kDa bands in 10-µl milk samples even days 1 and 2 of the start of lactation for milk of twice-transfected glands (see inset in Fig. 7). Although it is difficult to estimate LA-luc amounts quantitatively, we estimate that fusion protein production is greater than 0.6 ng of protein/ml milk (that is more than ~120 ng/milking) on days 4-5. ![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The data presented in this paper indicate that the mammary gland can be successfully transfected in vivo by means of insulin receptor-mediated endocytosis. Infusion of a transfecting construct into mammary gland seems more attractive than its injection into blood, since it allows a high local concentration of substance near the target cells to be achieved and minimizes a loss of delivering gene as a result of internalization by other cells possessing the same receptors. Taken together with the recent report of successful transformation of airway epithelium of rodents using receptor-mediated DNA uptake (12, 21), our data encourage further development of techniques to transform somatic cells in vivo by local administration rather than by intravenous injection. We show here that infusion of DNA-delivering construct into mammary ducts of mice or sheep can lead to luciferase activity in the tissue homogenates of mammary glands of these animals, implying that the mammary gland of mammals may turn out a suitable model organ for such experiments with a perspective to have an application in human gene therapy. Even more importantly, we demonstrate that it is possible to use this approach to effect the secretion of foreign proteins in milk.
The mammary gland of diary cattle can be regarded as a natural bioreactor for the production of secretable proteins and hormones of interest in milk. It is known that products of many eukaryotic genes require post-translational modifications for activity and therefore, their production with natural biological fluids in mammals is an attractive way to obtain many proteins for research and medical purposes. In addition, proteins derived in this manner are free from unwanted toxic contaminations that may exist when bacteria or yeast are used as a source for isolation of the products (45, 46). To get a protein secreted into milk, it is necessary to introduce the gene into the secretory cells of the mammary gland and to provide for excretion of the protein. Somatic gene transfer into the secretory cells of mammary glands, along with the creation of transgenic animals by conventional techniques, is a promising approach to achieve the above goal. Farm transgenic animals have the advantage of being able to retain appropriate genes over generations, but their derivation ab ovo is costly and requires years to obtain a result. In contrast, the transfection of somatic cells of adult organisms is a viable alternative to achieve a quick result without affecting the germ line.
As follows from the results presented, the secretory epithelium of the
mammary gland of mice and ewes transformed via receptor-mediated endocytosis is able not only to synthesize the product of interest faithfully but also to secrete it in milk. For this, we have used one
of the conventional reporter genes, luciferase, rather than any natural
milk protein or hormone. All standard reporter genes are antigenically
quite unrelated to any of mammalian proteins and thereby are suitable
to monitor foreign gene expression. However, all of them
(chloramphenicol acetyltransferase, -galactosidase, luciferase,
etc.) are not secretory proteins and, therefore, do not contain signals
to enter compartments of the exocytotic way. We have tested the signal
sequences from ovine
-lactoglobulin and the entire human LA protein
fused to the luciferase gene to obtain an active secretable enzyme. The
experiments on transformation of sheep mammary gland with the gene
encoding LA luciferase fusion protein have unambiguously demonstrated
the feasibility of producing proteins of interest in the milk of diary
cattle as a result of somatic transformation of epithelial cells of
mammary glands.
A characteristic of receptor-mediated gene transfer is the fact that DNA-delivering constructs, after surmounting of the cell surface, are caught within endosomes and thereby face a second potentially limiting barrier on the way to the cell nucleus. It has been shown that substances that induce an exit of the endosome content and, hence, rescue DNA from subsequent degradation in lysosomes are capable of increasing gene transfer efficiency (4, 5, 13, 25-27). Among them, adenoviruses are particularly effective. A replication-defective, E1a-deficient strain of human Ad5 and chicken embryo lethal orphan adenovirus have been reported to drastically augment the transferrin receptor-mediated delivery of constructs to cells in vitro (47). The former virus has been also successfully employed to transform in vivo airway epithelium of rodents (21). Apart from dl312 and chicken embryo lethal orphan adenoviruses, duck EDS-76 adenovirus has also been successfully used. The last two viruses have some advantage over the human, albeit defective strain, since in their natural form they are not able to penetrate mammalian cells. Here, we show that in vitro and in vivo transformation of mammary gland cells is greatly enhanced by co-transfection with adenoviruses. We found that although the effect of adenoviruses is pronounced at low amounts of administered DNA, the effect of adenoviruses is much lower or even absent at higher concentrations. Since the mean efficiency of in vivo transfection of mammary glands with virus-containing construct (3 nM DNA) was almost the same as for nonviral construct using a high DNA concentration (30 nM), we used the latter successfully to transfect sheep mammary glands. Although adenoviruses are most effective components for enhancement of gene transfer at the moment, they are only one of the possible approaches to bring about this enhancement, including amphipathic peptides (26, 27) and purified adenoviral protein components (48), and we intend to test some of these and other possibilities with respect to in vivo mammary gland transfection in the near future. Interestingly, overall positive charge of the transfection constructs (a lysine/nucleotide ratio near 2) appeared to provide significantly more efficient transfection of epithelial mammary cells, as measured by reporter gene expression, only in in vitro experiments; in vivo transfection of the mammary cells could be achieved only when the constructs had a ratio of less than 1. The basis of this difference is presently under investigation.
The expression of a foreign gene introduced into cells via receptor-mediated endocytosis, even for a prolonged period, is mostly accounted for by transient transformation (19). It seems quite probable that for future practice, continuous transformation for extended periods may also be useful. This would require a highly efficient integration of the foreign gene into the chromosomes of recipient cells after receptor-mediated uptake of exogenous DNA and its entry into the cell nucleus. Work in this direction is currently in progress.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. David A. Jans (Nuclear Signaling Laboratory, Division of Biochemistry and Molecular Biology, J. Curtin School of Medical Research, Australian National University, Canberra, Australia) for critical reading of the manuscript. We also gratefully acknowledge the assistance of Tatyana G. Kiseleva for the cell culture work and Drs. Ilyas N. Shaidullin and Igor Y. Shikhov (All-Russia Institute of Cattle Breeding, Dubrovitsy, Moscow Region) for the help with the work with ewes.
![]() |
FOOTNOTES |
---|
* This work was partially supported by Aeiveos Sciences Group LLC (Seattle, WA), Russian State Program "New Methods of Bioengineering; Genetic and Cell Engineering" Grants 342 and 150 and "National Priorities in Health Care and Medicine; 08, Gene Therapy" 08.01.01.04, and Russian Foundation for Basic Research Grant 97-04-50181.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Fax: 7-095-939-1115 or 7-095-977-0947; E-mail: asobol{at}1.biophys.bio.msu.ru.
1
The abbreviations used are: Ins-pLys,
insulin-polylysine conjugate; Ad5, human adenovirus type 5; EDS-76,
duck egg drop syndrome virus; LA, -lactalbumin; pLys,
poly-L-lysine; Str-pLys, streptavidin-polylysine conjugate;
CMV, cytomegalovirus; luc, luciferase; AU, arbitrary units.
2 Patents pending in U. S. A., Canada, Australia, Brazil, Japan, and 17 European countries.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|