基于RCAS-TVA技术的哺乳动物模型(英文)

Mammalian Models Based on RCAS-TVA Technique

NIU Yi-dong1,*, LIANG Shu-long2

(1. Laboratory Animal Center, Peking University People’s Hospital, Beijing 100044, China;

2. School of Basic Medical Sciences, Peking University, Beijing, 100083, China)

Abstract: The retroviral vector (RCAS) has been widely used in avian system to study development and diseases, but

is not suitable for mammals which do not produce the retrovirus receptor TVA. In this review, we trace the current uses of RCAS-TVA approach in mammalian system with improved strategies, including generation of tv-a transgenic mice, use of soluble TVA receptor and retroviral receptor-ligand fusion proteins, improvement of RCAS vectors, and compare a series of mammalian models in variant studies of gene function, development, oncogenesis and gene therapy. All those studies demonstrate that the RCAS-TVA based mammalian models are powerful tools for understanding the mechanisms and target treating of human diseases.

Key words: RCAS vector; TVA; Specific promoter; Transgenic animal; Mammalian model

基于RCAS-TVA技术的哺乳动物模型

牛屹东1,*，梁蜀龙2

（1．北京大学人民医院实验动物中心，北京 100044；2．北京大学基础医学院，北京 100083）

摘要：近年来，鸟类逆转录病毒载体（RCAS）及其受体（TVA）系统在哺乳动物转基因模型中得到广泛应用。本文对转tv-a基因小鼠的制备、特异性启动子选择、RCAS载体的改进等方面进行综述，展示近来RCAS-TVA系统在哺乳动物所取得的成果，并对RCAS-TVA基因转移技术的应用前景作一展望。

关键词：RCAS载体；TVA；特异性启动子；转基因动物；哺乳动物模型 中图分类号：Q812; Q78 文章标识码：A 文章编号：0254-5853-(2008)03-0335-011

Retroviruses are enveloped viruses possessing a

RNA genome, and replicate via a DNA intermediate. They rely on the enzyme, reverse transcriptase, to perform the reverse transcription of their genomes from RNA into DNA, which can then be integrated into the host's genome. The viruses then replicate as part of the cell's DNA (http://en.wikipedia.org/wiki/Retrovirus). So they have the ability to introduce new genetic information into the chromosomes of target cells, and serve as vehicles for transfer of exogenes (Orsulic, 2002). To date, retroviral vectors have been wildly developed to study gene function and therapy, developmental

Received date：2008-01-17； Accepted date：2008-03-28

processes, oncogenesis, and so forth (Logan & Tabin,

1998; Hu & Pathak, 2000; Barton & Medzhitov, 2002; Kawakami et al, 2003; Pao et al, 2003; Harpavat & Cepko, 2006; Du & Li, 2007). Among those retroviruses, the avian sarcoma-leukosis virus-A (ASLV-A)-derived vector called RCAS (Replication Competent ASLV long terminal repeat with Splice acceptor) is used most extensively in avian system, because high titer viral stocks can be harvested in avian cells without helper components. RCAS vectors are derived from the SR-A strain of Rous sarcoma virus (RSV) by deleting the src oncogene with a multi-cloning site where exogenes can

Foundation item: Supported by Peking University People’s Hospital Research and Development Foundation (RDB2007-03) 收稿日期：2008-01-17； 接受日期：2008-03-28

基金项目：北京大学人民医院研究与发展基金（RDB2007-03）

*

通讯作者（Corresponding author），Tel: 86-10-88325990, E-mail: niuyd07@yahoo.com.cn

336 Zoological Research Vol. 29

be inserted (Hughes & Kosik, 1984; Hughes et al, 1987; Petropoulos & Hughes, 1991; Boerkoel et al, 1993). The multi-cloning site can stably accommodate inserts up to 2.5 kb (Fig. 1). Expression of the inserted genes can be driven by either the viral long terminal repeat (LTR) or an appropriate internal promoter (Petropoulos et al, 1992; Du et al, 2006). A loss-of-function method, RCAS-RNAi (RNA interference) technique, has been verified to be efficient in “knocking down” the specific genes in avian developing craniofacial tissues, the limb bud, dorsal root ganglion, and the retina (Kawakami et al, 2003; Pekarik et al, 2003; Harpavat & Cepko, 2006).

Fig. 1 Constructs of the RSV and RCAS vector

The diagrams show the organization of viral DNA genomes and the location of genes (gag, pol, env, and src) and long terminal repeat (LTR). The src oncogene of RSV carries a splice acceptor (SA), which is retained in RCAS. The src oncogene has been deleted and replaced by a multi-cloning site in RCAS vector.

However, RCAS vectors can not be used in mammalian system directly without any improvement on the mammalian cells, which do not express the surface receptors for virus entry and infection. TVA, a member of the low-density-lipoprotein receptor family, is encoded by the tv-a gene and acts as the receptor for ASLV-A in avian cells (Bates et al, 1993; Young et al, 1993). mRNA transcribed from the tv-a gene is alternatively spliced to produce at least two proteins, a transmembrane and a GPI-anchored isoform (Bates et al, 1993). The mammalian cells are able to be infected and allow for genome integration by ASLV-A or RCAS virus if the cells are engineered to express TVA ectopically on the surface, and both of the isoforms are sufficient to permit infection of mammalian cells (Bates et al, 1993; Young et al, 1993).

Compared with the general mammalian counterparts, RCAS vectors can be constructed to encode all of the proteins required for assembly of infectious particles in addition to the transferred gene of interest, so they do not require helper cells (Hughes et al, 1987; Petropoulos & Hughes, 1991; Boerkoel et al, 1993). High-titer viral

stocks can be produced in avian cells (Himly et al, 1998; Schaefer-Klein et al, 1998). Viral proteins are inefficiently produced in mammalian cells, so the vectors can not spread from the target animals and cell-to-cell spreading within any individual is also prevented (Wills et al, 1989; Berberich et al, 1990). The lack of viral proteins also decreases the immune response by the host (Pinto et al, 2000). Furthermore, the most specific advantage of RCAS vectors is that multiple genes can be transferred sequentially into the target cells of a single transgenic animal (Federspiel et al, 1994; Holland et al, 1998; Murphy & Leavitt, 1999). This feature should be attributed to the sufficient supplement of TVA receptor which is not blocked by the poorly expressed viral envelope protein in mammalian cells. Recent experiments indicate that the RCAS vectors have the ability to infect non-dividing mammalian cells, including the primary neurons, although there is no direct evidence (Hatziioannou & Goff, 2001; Katz et al, 2002; Greger et al, 2004). The procedure to generate a RCAS-TVA based model is shown in Fig. 2.

Some potential limitations for using the RCAS-TVA system, however, should be given close attention. Target cells, tissues and organs must express the receptor TVA. Therefore, it is crucial to generate TVA transgenic animals before RCAS infection. RCAS can only accommodate an insert of less than 3 kb, but this limitation can be partially overcome by using pseudotyped vectors. The MLV (moloney murine leukemia virus), carrying capacity of insert up to 6-7 kb, has been efficiently pseudotyped with ASLV envelope protein (Soneoka et al, 1995; Murphy & Leavitt, 1999). But, integration site of viral DNA can not be controlled. Efficiency of infection is dependent on the accessibility of the organ and the proliferation rate of target cells. Description of various advantages and limitations of using the RCAS-TVA system has been reviewed in detail by Orsulic (2002).

1 Transgenic mammals expressing TVA molecules

1.1 Choice of specific promoters

The key element to produce tv-a transgenic animals is the tissue-specific and the lineage-specific promoters which decide specific expression of TVA in target cells, tissues and organs. Therefore, a nucleotide fragment

No. 3 NIU Yi-dong et al: Mammalian Models Based on RCAS-TVA Technique 337

consisting of the tv-a cDNA and a proper promoter must be constructed before RCAS infection. The specific promoters currently used to drive expression of tv-a gene in mammalian system are summarized in Tab. 1.

338 Zoological Research Vol. 29

Fig. 2 Schematic drawing of the RCAS-TVA technique in mammalian system

A: The target mammalian somatic cells are engineered to express TVA receptor under a tissue-specific promoter and therefore are susceptible to virus infection. B: Avian cells are transfected with a plasmid encoding the replication-competent, avian viral vector RCAS which contains the viral genes, gag, pol and env and a gene of interest (X gene). The viruses are produced in high titer and can infect avian cells again through the TVA receptors on the surface of cells. C: The mammalian cells expressing TVA are infected by RCAS vectors, and only the protein encoded by X gene is efficiently produced. Because very little viral proteins are produced, no infectious RCAS are replicated in mammalian cells. Therefore, the TVA receptors can be used repeatedly with different vectors. The neighbor cells can not be infected by RCAS because of their deficiency of TVA receptor. D: The target cells transfected with the genes of interest will show different destinies, such as proliferating or dying.

Tab. 1 Summary of the target cells, tissues and organs expressing TVA and

promoters used to drive expression of tv-a in mammalian system

Target Promoter/contexBone Brain

Hematopoietic cell Liver Lung

Mammary epithelial cell culture Mammary gland MEFs

Most or all tissues Neural crest cell Ovary Pancreas RK3E cell line

Skeletal muscle and heart Vascular endothelium

BSP GFAP nestin GP-Ibα albumin SPC MACT MMTV β-actin β-actin TRP2 β-actin , keratin elastase I CMV α-actin Tie2

Reference Li et al, 2005

Holland & Varmus, 1998; Yamashita et al, 2006 Holland et al, 1998 Murphy & Leavitt, 1999 Lewis et al, 2005 Fisher et al, 1999 Phillips et al, 2006 Du et al, 2006 Pao et al, 2003 Federspiel et al, 1996 Fisher et al., 1999

Orsulic et al, 2002; Xing & Orsulic, 2005 Kruse et al, 1993; Lewis et al, 2003 Fu et al, 2005 Federspiel et al, 1994 Montaner et al, 2003

BSP, bone sialoprotein; CMV, cytomegalovirus; GFAP, glial fibrillary acidic protein; GP, glycoprotein; MACT, mouse β-actin; MEFs, murine embryonic fibroblasts; MMTV, mouse mammary tumor virus; RK3E, rat kidney epithelial cell line; SPC, surfactant protein-C; TRP2, tyrosinase-related protein-2.

Additionally, numerous studies have indicated that some other promoters, including the ovary specific promoter (OSP1) and the high-affinity folate receptors promoter (HAFR) (Godwin et al, 1995; Goldsmith et al,

No. 3 NIU Yi-dong et al: Mammalian Models Based on RCAS-TVA Technique 339

1999), the modified rat probasin (rPB) promoters (Furuhata et al, 2003), and the neuroactive peptide cholecystokinin (CCK) promoter (Chhatwal et al, 2007), are of potential value for tissue specific expression of the tv-a gene.

1.2 Both isoforms of TVA molecule are sufficient for

acceptance of the RCAS vectors

As mentioned earlier, two isoforms, a 1999; Fisher et al, 1999).

However, the microinjection method has some potential limitations: 1. limited success in producing transgenic animals of larger species; 2. requirement of special equipment for DNA microinjection and high technical skills; 3. labor intensive. The new mean has been developed by using sperm cells, including spermatogonia, as the vehicle to deliver exogenous DNA transmembrane and a GPI-linked one, have been identified. In the current tv-a transgenic mice, the GPI-anchored isoform is commonly used (Federspiel et al, 1996; Holland et al, 1998; Du et al, 2006), but the transmembrane isoform has also be used successfully in the study of Murphy & Leavitt (1999). Therefore, both isoforms can accept the RCAS vectors although the physiological functions have not been determined. 1.3 Gene transfer methods

The most widely used method to generate transgenic animals is to microinject foreign DNA into the pronucleus of a fertilized egg. Pronuclear microinjection is conceptually straightforward, although it demands special equipment and technical skill, and has the additional feature that any cloned DNA can be used (Palmiter & Brinster, 1986). The primary mammalian model based on the RCAS-TVA approach was developed in transgenic mice expressing TVA specifically in muscle cells. They were generated by microinjecting a nucleotide fragment consisting of the tv-a cDNA and chicken α-actin promoter fragment into fertilized mice eggs (Federspiel et al, 1994). Subsequently, Holland et al. generated mice expressing TVA on the surface of glial cells by microinjection of Gtv-a transgene, which is a 2.2 kb fragment of the GFAP promoter driving expression of the quail tv-a cDNA and a fragment from the mouse protamine gene (MP-1) supplying an intron and signal for polyadenylation (Holland et al, 1998, 2000). Using the microinjection method, then, tv-a transgenic mice were extensively created to accept RCAS vectors in cancer models of mammary, ovary, pancreas, liver, lung, brain, vascular endothelium, melanoma, and other cell types (Korfhagen et al, 1990; Holland & Varmus, 1998; Holland et al, 1998; Fisher et al, 1999; Orsulic et al,2002; Montaner et al, 2003; Lewis et al, 2003, 2005; Pao et al, 2003; Fu et al, 2005; Du et al, 2006) and development models, including neuron, hemapoietic cell lines, and other organs (Doetsch et al, 1999; Murphy & Leavitt, into oocytes, and is therefore called “sperm-mediated gene transfer” (SMGT, Lavitrano et al, 1989). Based on SMGT, transgenic rats (Hamra et al, 2002; Orwig et al, 2002), pigs (Honaramooz et al, 2002) and goats (Honaramooz et al, 2003) have now been produced. As the improved method of SMGT, testis-mediated gene transfer (TMGT) has been demonstrated to be practical in delivering foreign DNA directly into the interstitial space of adult mammalian testes (Fig. 3A), and then the exogenous DNA is transmitted to oocytes via fertilization (Sato et al, 1999; Sato & Nakamura, 2004). Recently, He et al (2006) further indicated that transgenic efficiency of TMGT was very high in both F1 and F2 mice offspring (41% and 37% respectively), and that TMGT was suitable for creating transgenic animals. The TMGT technique is very simple and convenient. A needle, a plastic disposal syringe, and a dissecting microscope are sufficient for delivery of DNA. The TMGT technique opens a new perspective for generating tv-a transgenic mammals (Fig. 3B), although it requires further improvement.

More recently, Yang et al (2007) established a rapid procedure for obtaining transgenic mice by directly injecting exogenes into the ovaries of fertile mice, called ovary mediated gene transfer (OMGT). After natural fertilization, healthy transgenic mice were obtained, and the introduced foreign gene was inherited by F1 offspring (64.9%) and transmitted to F2 progeny (66.94%) stably. The foreign gene was found to be not only integrated into the genome with a high frequency of 85.71% (multiple site versus single site insertions analyzed by FISH), but also translated into a functional protein and transferred to the next generation. Although the procedure is somewhat more complicated than TMGT, OMGT is still a useful technique with a much higher success rate for creating transgenic mammals via efficient and functional integration of the foreign gene into the host genome and stable transmission of the

340 Zoological Research Vol. 29

Fig. 3 Schematic drawing of testis-mediated gene transfer (TMGT) method and retroviral gene delivery to mammals in vivo

A: Injection of solution containing tv-a gene driven by tissue-specific promoter and liposomes (in some cases) is performed at the corner of the testis near the caput epididymis to a depth of 5-6mm (Sato et al, 1999). B: The adult male mice carrying tv-a gene driven by a tissue-specific promoter are mated with normal females，and the promoter-tv-a sequence will be delivered into zygotes by sperms. The offspring (F1 generations) are examined, and only those expressing TVA in specific tissues or organs are left for retroviral infection. Chicken DF-1 cells transfected with RCAS vectors carrying genes of interest are propagated to obtain high titer viruses. Producer DF-1 cells, cell supernatants, or concentrated viruses can be used to infect TVA-expressing mice. Tissue-specific infection can be achieved by direct injection of viruses or virus-producing cells into an organ in which TVA is expressed. The anatomy atlas of testis is modified from the website http://msjensen.cehd.umn.edu/Webanatomy/image_database/Reproductive/testis2.gif.

foreign gene to the offspring. Simplicity of procedure and cost-effectiveness are the advantages of OMGT used for tv-a transgenic animals in contrast to other traditional methods, such as pronuclear microinjection.

2 RCAS vectors used for mammalian sys- tem

2.1 Modified vectors for overcoming the limited in-

sertion size

No. 3 NIU Yi-dong et al: Mammalian Models Based on RCAS-TVA Technique 341

As mentioned earlier, RCAS can only accommodate an insert of less than 3 kb. This may not be a significant problem because most cDNAs studied are less than 2.5 kb (Fisher et al, 1999). This limitation can, however, be overcome by using RCAS and ASLV-A Env pseudotyped HIV and MLV vectors, respectively (Murphy & Leavitt, 1999; Lewis et al, 2001), even if more than 3 kb sequences must be inserted.

2.2 Improved vectors for infection of a broad range

of cell types

Although some reports have shown that RCAS can infect non-dividing cells (Lu et al, 1999; Hatziioannou & Goff, 2001), low efficiency suggests entrance of viral DNA into nucleus depends on mitosis of host cells. To overcome this limitation, Lewis et al (2001) succeeded in using a pseudotyped replication-deficient HIV-1 based lentiviral vector to infect non-dividing TVA positive cells. However, there is no evidence that can clarify the efficiency of this vector in vivo. Therefore, further studies are required to improve the ability of RCAS vectors into the genome of non-dividing host cells. 2.3 Vectors used in mammalian system

The RCAS family consists of a group of vectors for variant demand. Actually, the current vectors used in mammalian system, have an env gene from a murine retrovirus instead of one from the ASLV. These vectors are named as RCASBP, in which env gene is derived from an amphotropic virus or ecotropic virus. The properties of RCASBP vectors are summarized on the website:

http://home.ncifcrf.gov/hivdrp/RCAS/tables.html#table2.

3 Overview of current mammalian models

based on RCAS-TVA technique

The primary mammalian model based on RCAS-TVA approach was developed in mice (Mus musculus) by Federspiel et al (1994). This work opens a new way to study development and oncogenesis, and sheds light on models for tissue-specific gene therapy. 3.1 Use of RCAS-TVA based models to study

developmental processes

RCAS-TVA based method has been proven to be useful in developmental studies in mammalian system. Murphy & Leavitt (1999) used the GP-Ibα regulatory sequences to achieve megakaryocyte-lineage of mice restricted expression of TVA. They infected the cells with RCAS-PURO (expresses puromycin-resistance gene) and RCAS-AP (expresses human placental alkaline phosphatase) in vitro and in vivo, then generated and characterized a pure population of primary CD41-positi- ve megakaryocyte progenitors. The in vitro study indicated that IL-3 inhibits the development of mature megakaryocytes. Doetsch et al (1999) infected SVZ (subventricular zone) astrocytes of tv-a transgenic mice with RCAS-AP in vivo, and the AP-positive cells were examined and traced. They demonstrated that SVZ astrocytes act as neural stem cells in normal brain. Study of lung development has been reported using the RCAS-TVA model (Fisher et al, 1999). Lung buds of the SPC-tv-a transgenic mice were infected with different RCAS viruses to study the effects on branching morphology in vitro. To study bone development in vivo, Li et al (2005) established the BSP-tv-a transgenic mice which selectively expressed TVA in skeletal tissues. After infecting with RCASBP-Cbfa1/Runx2, bone and tooth formation was delayed. They validated this model as a unique system for studying molecular events associated with bone formation in vivo.

Dunn et al (2000, 2001)have infected the neural precursor cells and the melanoblasts expressing TVA driven by nestin and Dopachrome tautomerase promoter (DCT) with RCAS-Wnt, RCAS-lacZ (β-galactosidase) and RCAS-Tyr (tyrosinase) respectively in primary culture and in utero. They demonstrated that the RCAS-TVA method was useful to study the development of neural systems. Recently, the RCAS-TVA system was successfully adapted by Yamashita and colleagues to study neurogenesis in vivo (Yamashita et al, 2006). They

traced maturation of neurons by infecting the GFAP tv-a

(Gtv-a) transgenic mice with RCAS-EGFP

(CAG-CAT-enhanced green fluorescent protein), and indicated that SVZ-derived neuroblasts differentiated into mature neurons in the post-stroke striatum. 3.2 Use of RCAS-TVA based models to study onco- genesis

Currently, most models of tumors are traditionally germ-line models constructed by transgenic or knockout approaches. The major limitation of these models is that the initiation and progression of carcinogenesis can not be understood. However, the RCAS-TVA method overcomes this limitation and allows investigation of the carcinogenic potential of candidate oncogenes in somatic

342 Zoological Research Vol. 29

cells in vivo without creating individual transgenic lines (Du & Li, 2007). To date, several oncogenes have been studied in murine system using this technique. The variant cancers or tumors and oncogenes studied are summarized in Tab. 2.

Cancer is thought to be associated with multiple genetic alterations. Microarray analysis of ovarian cancer has demonstrated that oncogenesis of ovarian neoplasms is controlled by many genes, and that changes in expression of these genes correlate with malignancy potential (Warrenfeltz et al, 2004). To study the effect of multiple genes on carcinogenesis, the RCAS-TVA system provides a flexible method to deliver several genes simultaneously or sequentially. Holland et al (2000) infected the Ntv-a transgenic mice with a combination of DF-1 cells infected with and producing RCAS-Ras and RCAS-Akt. They found that combination of activated Ras and Akt induces high grade gliomas with the histological features of human glioblastoma multiformes (GBMs) although neither activated Ras nor Akt alone is sufficient to induce GBM formation.

Orsulic et al (2002) isolated ovarian cells from TVA transgenic mice deficient for p53, and infected the target cells with RCAS-Myc, RCAS-Ras, and RCAS-Akt. Their study showed that addition of any two of the oncogenes Myc, Ras, and Akt were sufficient to induce ovarian tumor formation when infected cells were injected into the recipient mice at subcutaneous, intraperitoneal, or ovarian sites. They demonstrated that the ovarian surface epithelium is the precursor tissue for these ovarian carcinomas, and that introduction of oncogenes causes phenotypic changes in the ovarian surface epithelial cells.

A mouse model for hepatocellular carcinoma was generated by infecting tv-a transgenic wild-type and p53 null mice with RCAS-PyMT (Lewis et al, 2005). Tumors were induced in both wild-type and p53 null mice, but only in the mice lacking an intact p53 gene the resulting tumors were poorly differentiated, invasive, and metastatic to the lungs. This study demonstrates that metastasis is dependent on both the oncogene and the absence of p53.

3.3 Use of RCAS-TVA based models to study gene

function

The lost-of-function and “knock out” techniques are robust and practical for studying gene function in mammalian system. The retroviral vectors have been validated to express short hairpin RNA (shRNA) under the control of an RNA polymerase III promoter for the purpose of inhibiting gene expression in a sequence- specific manner (Brummelkamp et al, 2002; Hemann et al, 2003; Rubinson et al, 2003).

To date, RNA interference (RNAi) technique has been incorporated successfully with the RCAS-TVA method to study gene function in avian development. Bron et al (2004) knocked the neuropilin-1 (Nrp-1) receptor in chick embryos using the RCAS-RNAi technique. They found that Sema3A-induced growth cone was inhibited in dorsal root ganglion (DRG) neurons. This result demonstrated the functional knockdown of Nrp-1. Harpava and Cepko (2006) delivered hairpins mediating RNA interference to the

Tab. 2 Cancers/tumors and oncogenes studied in murine system by RCAS-TVA approach

Cancer/tumor Oncogene Target Breast cancer Liver cancer

Nervous system tumors Astrocytoma Glioblastoma Oligoastrocytoma Oligodendroglioma tumors (PNETs) Ovarian cancer

Myc, Ras, Akt

Ovarian cells in culture

Ovarian cancer cell lines and tumors with defined genetic alterations

Pancreatic cancer

PyMT, Myc

Pancreas in vivo

Lewis et al, 2003 Orsulic et al, 2002 Xing & Orsulic, 2005

Cre PyMT, Neu PyMT, Myc Ras, Akt PDGF-B

Mammary gland in vivo

Liver parenchyma in vivo

Brain in vivo

Brain in vivo, Primary brain cell cultures

Neural progenitor cells, brain in vivo

Reference Fisher et al, 1999 Du et al, 2006 Lewis et al, 2005

Holland & Varmus, 1998 Holland et al, 2000

Holland & Varmus, 1998; Dai et al, 2001 Dai et al, 2001 Fults et al, 2002

Primitive neuroectodermal Myc

PDGF-B, platelet derived growth factor-b chain.

No. 3 NIU Yi-dong et al: Mammalian Models Based on RCAS-TVA Technique 343

developing chick eye by RCAS viruses. They 'knocked down' specific genes in infected areas of the retina. The knock down persisted as the retina matured and could be detected using in situ hybridization. Furthermore, the amount of retinal tissue affected could be controlled by manipulating the degree of infection.

In mammalian system, Bromberg-White et al (2004) created a RCAS vector capable of expressing shRNA that inhibits the expression of glyceraldehydes-3-phos- phate dehydrogenase (GAPDH) gene, and reduces GAPDH expression in cell line A375. They demonstrated that RCAS vectors can be used to stably express shRNA to inhibit gene expression in loss-of-function analyses of specific genes in vitro as well as in vivo.

The RCASBP-Y vector has been modified to incorporate “Gateway” site-specific recombination cloning of genes into the viral construct, and will allow for the efficient transfer and expression of cDNAs required for functional genomic analyses in both avian and mammalian model systems (Loftus et al, 2001).

3.4 Use of soluble TVA receptor, TVA-ligand bridge

proteins and RCAS system for gene therapy RCAS system has been considered useful for gene therapy of cancers (Orsulic, 2002; Xing & Orsulic, 2005). However, gene therapy is dependant on the ability of target cancer cells to accept the viral vectors carrying therapeautic or suicide genes. Many TVA receptor transgenic models have been generated in mammalian system to accept RCAS vectors, simultaneously new methods are developed. Several studies have indicated that the membrane TVA receptor is not an absolute requirement for virus infection, and RCAS vectors linked with a soluble TVA can be delivered into receptor-deficient cells (Snitkovsky & Young, 1998; Damico & Bates, 2000; Contreras-Alcantara et al, 2006). The viral receptor function of TVA is determined by a 40-residue, cysteine-rich motif called the LDL-A module, which is highly homologous to the human low-density lipoprotein receptor (LDLR) ligand-binding repeats (LDL-A modules). It has been demonstrated that the LDL-A module of TVA is necessary and sufficient to mediate efficient EnvA binding and ASLV-A infection (Rong & Bates, 1995). Therefore, the soluble TVA receptor is an ideal candidate for transferring RCAS to target cells deficient in membrane TVA.

Additionally, several proteins consisting of a TVA

receptor-ligand fusion structure have been developed to serve as bifunctional bridge to surface receptors of target cells and RCAS vectors (Fig. 4). The bridge proteins contain the extracellular domain of TVA and a peptide which can bind to surface receptors of target cells (Orsulic, 2002). Several authors have succeeded in infecting the mammalian cells expressing cognate cellular receptors using the bridge proteins consisting of the domain of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), or heregulin (Boerger et al, 1999; Snitkovsky et al, 2000, 2001; Snitkovsky & Young, 1998, 2002). This method provides a flexible way to target entry of RCAS vectors to mammalian cells.

Fig. 4 Delivery of RCAS vectors into target mammalian cells by retroviral receptor-ligand fusion proteins

The fusion protein is comprised of the extracellular domain of TVA fused with a ligand protein which permits it to bind to RCAS viral ENV and to cell surface receptor respectively.

Hu et al (2007) investigated for the first time the characteristics of RCAS as an alternative vector system for transduction of hematopoietic stem and progenitor cells. The new vectors were modified by replacing the avian env gene with the gene encoding amphotropic or ecotropic murine Env protein, which allows RCAS vectors to infect mammalian cells efficiently (Barsov & Hughes, 1996; Barsov et al, 2001). They used nonhuman primate autologous transplantation models to test whether the RCAS vectors can efficiently transduce rhesus macaque CD34+ hematopoietic stem and progenitor cells. This study showed that RCAS vectors could efficiently and stably transduce the CD34+ hematopoietic progenitor cells with an efficiency of transduction of up to 34% in vitro, and that highly polyclonal hematopoietic reconstitution in myeloid and lymphoid lineages was observed up to 18 months post-transplantation in animals transplanted with RCAS vector-transduced autologous CD34+ cells. Hu et al

344 Zoological Research Vol. 29

(2007) indicated that the RCAS system should be explored and further optimized for gene therapy applications targeting hematopoietic stem and progenitor cells.

4 Conclusion and future research

This review shows that the RCAS-TVA based technique is very useful and valid in various areas, including basic biology, medicine and clinical research. To date, a large number of RCAS vectors (and ancillary tools, including soluble receptors, receptor-ligand fusion proteins, mammalian cell lines expressing receptors, and tv-a transgenic mice) have been developed for mammalian system, and the number of mammalian models (especially the mouse models) is constantly increasing. These models can be used for a number of different study purposes: gene function, development, carcinogenesis and gene therapy. Bioreactor is also a novel potential for the use of RCAS-TVA system in the future. Pronuclear microinjection is the major method today for the production of transgenic animal bioreactor, but repeated operation is inevitable to create different transgenic animals producing different bioactive proteins. The RCAS-TVA technique provides a convenient and flexible way to produce a variety of biological products in one transgenic animal, in which different target genes can be introduced to the tissues or organs expressing TVA

References:

Barsov EV, Hughes SH. 1996. Gene transfer into mammalian cells by a

Rous sarcoma virus-based retroviral vector with the host range of the amphotropic murine leukemia virus [J]. J Virol, 70: 3922-3929. Barsov EV, Payne WS, Hughes SH. 2001. Adaptation of chimeric

retroviruses in vitro and in vivo: Isolation of avian retroviral vectors with extended host range [J]. J Virol, 75: 4973-4983.

Barton GM, Medzhitov R. 2002. Retroviral delivery of small

interfering RNA into primary cells [J]. Proc Natl Acad Sci USA, 99: 14943-14945.

Bates P, Young JA, Varmus HE. 1993. A receptor for subgroup a Rous

sarcoma virus is related to the low density lipoprotein receptor [J]. Cell, 74: 1043–1051.

Berberich SL, Macias M, Zhang L, Turek LP, Stoltzfus CM. 1990.

Comparison of Rous sarcoma virus RNA processing in chicken and mouse fibroblasts: evidence for double-spliced RNA in nonpermissive mouse cells [J]. J Virol, 64: 4313-4320.

Boerger AL, Snitkovsky S, Young JAT. 1999. Retroviral vectors

preloaded with a viral receptor-ligand bridge protein are targeted to specific cell types [J]. Proc Natl Acad Sci USA, 96: 9867-9872. Boerkoel CF, Federspiel MJ, Salter DW, Payne W, Crittenden LB,

molecules simultaneously, sequentially or repeatedly. For the objective of producing pharmaceuticals for treating human diseases, mammary glands may be the ideal tissue for use of the RCAS-TVA system.

The TMGT and OMGT techniques open a new

perspective in creating tv-a transgenic mammals.

Compared with the conventional pronuclear microinjec- tion approach, use of TMGT and OMGT is much cheaper and faster with quick and effective delivery of genes to target tissues (Sato, 2005; Yang et al, 2007). Therefore, the cost for the whole experiment is decreased. The current studies demonstrate that gene knock down can be made in vitro and in vivo by using the RCAS system expressing RNAi. It can be predicted that the RCAS-RNAi technique will be very useful for gene function (or loss-of-function) studies. To improve the infectious ability of RCAS vectors to cells absent of mitosis, more and more vectors are being developed.

Furthermore, novel uses of the RCAS-TVA system including infection of non-dividing somatic cells, and neurons, are being developed in mammalian models. On the whole, the RCAS-TVA based mammalian model is a powerful tool for understanding the mechanism and target treatment of human diseases. We anticipate that new uses for the RCAS-TVA method in mammals will be developed with a better understanding of retroviruses, its hosts, and using other new biological techniques. Kung HJ, Hughes SH. 1993. A new defective retroviral vector system based on the Bryan strain of Rous sarcoma virus [J]. Virology, 195: 669-679.

Bromberg-White JL, Webb CP, Patacsil VS, Miranti CK, Williams BO,

Holmen SL. 2004. Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector [J]. J Virol, 78: 4914-4916.

Bron R, Eickholt BJ, Vermeren M, Fragale N, Cohen J. 2004.

Functional knockdown of neuropilin-1 in the developing chick nervous system by siRNA hairpins phenocopies genetic ablation in the mouse [J]. Dev Dyn, 230: 299-308.

Brummelkamp TR, Bernards R, Agami R. 2002. Stable suppression of

tumorigenicity by virus-mediated RNA interference [J]. Cancer Cell, 2: 243-247.

Contreras-Alcantara S, Godby JA, Delos SE. 2006. The single ligand

binding repeat of TVA, a low density lipoprotein receptor related protein, contains two ligand binding surfaces [J]. J Biol Chem, 281: 22827-22838.

Chhatwal JP, Hammack SE, Jasnow AM, Rainnie DG, Ressler KJ. 2007.

Identification of cell-type-specific promoters within the brain using lentiviral vectors [J]. Gene Ther, 14: 575-583.

No. 3 NIU Yi-dong et al: Mammalian Models Based on RCAS-TVA Technique 345

Dai C, Celestino JC, Okada Y, Louis DN, Fuller GN, Holland EC. 2001.

PDGF autocrine stimulation dedifferentiates cultured astrocytes and induces oligodendrogliomas and oligoastrocytomas from neural progenitors and astrocytes in vivo [J]. Genes Dev, 15: 1913-1925.

Damico R, Bates P. 2000. Soluble receptor-induced retroviral infection

of receptor-deficient cells [J]. J Virol, 74: 6469-6475.

Doetsch F, Caillé I, Lim DA, García-Verdugo JM, Alvarez-Buylla A.

1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain [J]. Cell, 97: 703-716.

Du Z, Li Y. 2007. RCAS-TVA in the mammary gland: an in vivo

oncogene screen and a high fidelity model for breast transformation? [J]. Cell Cycle, 6: 823-826.

Du Z, Podsypanina K, Huang S, McGrath A, Toneff MJ, Bogoslovskaia

E, Zhang X, Moraes RC, Fluck M, Craig Allred DC, Lewis MT, Varmus HE, Li Y. 2006. Introduction of oncogenes into mammary glands in vivo with an avian retroviral vector initiates and promotes carcinogenesis in mouse models [J]. Proc Natl Acad Sci USA, 103: 17396-17401.

Dunn KJ, Incao A, Watkins-Chow D, Li Y, Pavan WJ. 2001. In utero

complementation of a neural crest-derived melanocyte defect using cell directed gene transfer [J]. Genesis, 30: 70-76.

Dunn KJ, Williams BO, Li Y, Pavan WJ. 2000. Neural crest-directed

gene transfer demonstrates Wnt1 role in melanocyte expansion and differentiation during mouse development [J]. Proc Natl Acad Sci USA, 97: 10050-10055.

Federspiel MJ, Bates P, Young JAT, Varmus HE, Hughes SH. 1994. A

system for tissue-specific gene targeting: Transgenic mice susceptible to subgroup A avian leukosis virus-based retroviral vectors [J]. Proc Natl Acad Sci USA, 91: 11241-11245.

Federspiel MJ, Swing DA, Eagleson B, Reid SW, Hughes SH. 1996.

Expression of transduced genes in mice generated by infecting blastocysts with avian leukosis virus-based retroviral vectors [J]. Proc Natl Acad Sci USA, 93: 4931-4936.

Fisher GH, Orsulic S, Holland E, Hively WP, Li Y, Lewis BC, Williams

BO, Varmus HE. 1999. Development of a flexible and specific gene delivery system for production of murine tumor models [J]. Oncogene, 18: 5253-5260.

Fu SL, Huang YJ, Liang FP, Huang YF, Chuang CF, Wang SW, Yao JW.

2005. Malignant transformation of an epithelial cell by v-Src via tv-a-mediated retroviral infection: A new cell model for studying carcinogenesis [J]. Biochem Biophys Res Commun, 338: 830-838. Fults D, Pedone C, Dai C, Holland EC. 2002. MYC expression

promotes the proliferation of neural progenitor cells in culture and in vivo [J]. Neoplasia, 4: 32-39.

Furuhata S, Ide H, Miura Y, Yoshida T, Aoki K. 2003. Development of

a prostate-specific promoter for gene therapy against androgen-independent prostate cancer [J]. Mol Ther, 7: 366-374. Godwin AK, Miller PD, Getts LA, Jakson K, Sonoda G, Schray KJ,

Testa JR, Hamilton TC. 1995. Retroviral-like sequences specifically expressed in the rat ovary detect genetic differences between normal and transformed rat ovarian epithelial cells [J]. Endocrinology, 136: 4640-4649.

Goldsmith ME, Short KJ, Elwood PC, Kowan KH. 1999. A

recombinant adenoviral vector with selective transgene expression in ovarian cancer cells [J]. Proc Am Assoc Cancer Res, 40: 479. Greger JG, Katz RA, Taganov K, Rall GF, Skalka AM. 2004.

Transduction of terminally differentiated neurons by avian sarcoma virus [J]. J Virol, 78: 4902-4906.

Hamra FK, Gatlin J, Chapman KM, Grellhesl DM, Garcia JV, Hammer

RE, Garbers DL. 2002. Production of transgenic rats by lentiviral transduction of male germ-line stem cells [J]. Proc Natl Acad Sci USA, 99: 14931-14936.

Harpavat S, Cepko CL. 2006. RCAS-RNAi: a loss-of-function method

for the developing chick retina [J]. BMC Dev Biol, 6: 2 (http://www.biomedcentral.com/1471-213X/6/2).

Hatziioannou T, Goff SP. 2001. Infection of nondividing cells by Rous sarcoma virus [J]. J Virol, 75: 9526-9531.

He X, Qi B, Liu GS, Yu WD, Chen QX. 2006. A novel method to

transfer gene in vivo system [J]. Prog Biochem Biophys, 33: 685-690.

Hemann MT, Fridman JS, Zilfou JT, Hernando E, Paddison PJ,

Cordon-Cardo C, Hannon GJ, Lowe SW. 2003. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo [J]. Nat Genet, 33: 396-400.

Himly M, Foster DN, Bottoli I, Iacovoni JS, Vogt PK. 1998. The DF-1

chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses [J]. Virology, 248: 295-304.

Holland EC, Celestino J, Dai C, Schaefer L, Sawaya RE, Fuller GN.

2000. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice [J]. Nat Genet, 25: 55-57. Holland EC, Hively WP, DePinho RA, Varmus HE. 1998. A

constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice [J]. Genes Dev, 12: 3675-3685.

Holland EC, Varmus HE. 1998. Basic fibroblast growth factor induces

cell migration and proliferation after glia-specific gene transfer in mice [J]. Proc Natl Acad Sci USA, 95: 1218-1223.

Honaramooz A, Megee SO, Dobrinski I. 2002. Germ cell

transplantation in pigs [J]. Biol Reprod, 66: 21-28.

Honaramooz A, Behboodi E, Blash S, Megee SO, Dobrinski I. 2003.

Germ cell transplantation in goats [J]. Mol Reprod Dev, 64: 422-428.

Hu J, Ferris A, Larochelle A, Krouse A, Metzger ME, Donahue RE,

Hughes SH, Dunbar CE. 2007. Transduction of rhesus macaque hematopoietic stem and progenitor cells with avian sarcoma and leukosis viral vectors [J]. Hum Gene Ther, 18: 691-700.

Hu WS, Pathak VK. 2000. Design of retroviral vectors and helper cells

for gene therapy [J]. Pharm Rev, 52: 493-512.

Hughes SH, Greenhouse JJ, Petropoulos CJ, Sutrave P. 1987. Adaptor

plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors [J]. J Virol, 61: 3004-3012. Hughes ST, Kosik E. 1984. Mutagenesis of the region between env and

src of the SR-A strain of Rous sarcoma virus for the purpose of constructing helper-independent vectors [J]. Virology, 136: 89-99. Katz RA, Greger JG, Darby K, Boimel P, Rall GF, Skalka AM. 2002.

Transduction of interphase cells by avian sarcoma virus [J]. J Virol, 76: 5422-5434.

Kawakami Y, Rodríguez-León J, Koth CM, Büscher D, Itoh T, Raya Á,

Ng JK, Esteban CR, Takahashi S, Henrique D, Schwarz MF, Asahara H, Belmonte JCI. 2003. MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb [J]. Nat Cell Biol, 5: 513-519.

Korfhagen TR, Glasser SW, Wert SE, Bruno MD, Daugherty CC,

McNeish JD, Stock JL, Potter SS, Whitsett JA. 1990. Cis-acting sequences from a human surfactant protein gene confer pulmonary- specific gene expression in transgenic mice [J]. Proc Natl Acad Sci USA, 87: 6122-6126.

Kruse F, Rose SD, Swift GH, Hammer RE, MacDonald RJ. 1993. An

endocrine-specific element is an integral component of an exocrine-specific pancreatic enhancer [J]. Genes Dev, 7: 774-786. Lavitrano M, Camaioni A, Fazio VM, Dolci S, Farace MG, Spadafora

C. 1989. Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice [J]. Cell, 57: 717-723.

Lewis BC, Chinnasamy N, Morgan RA, Varmus HE. 2001.

Development of an avian leukosis sarcoma virus subgroup A pseudotyped lentiviral vector [J]. J Virol, 75: 9339-9344.

Lewis BC, Klimstra DS, Socci ND, Xu S, Koutcher JA, Varmus HE.

2005. The absence of p53 promotes metastasis in a novel somatic mouse model for hepatocellular carcinoma [J]. Mol Cell Biol, 25: 1228-1237.

Lewis BC, Klimstra DS, Varmus HE. 2003. The c-myc and PyMT

oncogenes induce different tumor types in a somatic mouse model

346 Zoological Research Vol. 29

for pancreatic cancer [J]. Genes Dev, 17: 3127-3138.

Li L, Zhu J, Tu Q, Yamauchi M, Sodek J, Karsenty G, Tang J, Chen J.

2005. An in vivo model to study osteogenic gene regulation: targeting an avian retroviral receptor (TVA) to bone with bone sialoprotein (BSP) promoter [J]. J Bone Min Res, 20: 1403-1413. Loftus SK, Larson DM, Watkins-Chow D, Church DM, Pavan WJ.

2001. Generation of RCAS vectors useful for functional genomic analyses [J]. DNA Res, 8: 221-226.

Logan M, Tabin C. 1998. Targeted gene misexpression in chick limb

buds using avian replication-competent retroviruses [J]. Methods, 14: 407-420.

Lu Q, Gore M, Zhang Q, Camenisch T, Boast S, Casagranda F, Lai C,

Skinner MK, Klein R, Matsushima GK, Earp HS, Goff SP, Lemke G. 1999. Tyro-3 family receptors are essential regulators of mammalian spermatogenesis [J]. Nature, 398: 723-728.

Montaner S, Sodhi A, Molinolo A, Bugge TH, Sawai ET, He Y, Li Y,

Ray PE, Gutkind JS. 2003. Endothelial infection with KSHV genes in vivo reveals that vGPCR initiates Kaposi's sarcomagenesis and can promote the tumorigenic potential of viral latent genes [J]. Cancer Cell, 3: 23-36.

Murphy GJ, Leavitt AD. 1999. A model for studying megakaryocyte

development and biology [J]. Proc Natl Acad Sci USA, 96: 3065-3070.

Orsulic S. 2002. An RCAS-TVA-based approach to designer mouse

models [J]. Mam Gen, 13: 543-547.

Orsulic S, Li Y, Soslow RA, Vitale-Cross LA, Gutkind JS, Varmus HE.

2002. Induction of ovarian cancer by defined multiple genetic changes in a mouse model system [J]. Cancer Cell, 1: 53-62.

Orwig KE, Avarbock MR, Brinster RL. 2002. Retrovirus-mediated

modification of male germline stem cells in rats [J]. Biol Reprod, 67: 874-879.

Palmiter RD, Brinster RL. 1986. Germ-line transformation of mice [J].

Annu Rev Genet, 20: 465-499.

Pao W, Klimstra DS, Fisher GH, Varmus HE. 2003. Use of avian

retroviral vectors to introduce transcriptional regulators into mammalian cells for analyses of tumor maintenance [J]. Proc Natl Acad Sci USA, 100: 8764-8769.

Pekarik V, Bourikas D, Miglino N, Joset P, Preiswerk S, Stoeckli ET.

2003. Screening for gene function in chicken embryo using RNAi and electroporation [J]. Nat Biotechnol, 21: 93-96.

Petropoulos CJ, Hughes SH. 1991. Replication-competent retroviral

vectors for the transfer and expression of gene cassettes in avian cells [J]. J Virol, 65: 3728-3737.

Petropoulos CJ, Payne W, Salter DW, Hughes SH. 1992. Using avian

retroviral vectors for gene transfer [J]. J Virol, 66: 3391-3397.

Phillips PM, Wynn PC, Sheehy PA. 2006. Successful adaptation of the

RCAS-TVA gene transfer system for use in a bovine mammary epithelial cell culture model [J]. Mol Ther, 13: S318.

Pinto VB, Prasad S, Yewdell J, Bennink J, Hughes SH. 2000.

Restricting expression prolongs expression of foreign genes introduced into animals by retroviruses [J]. J Virol, 74: 10202-10206.

Rong L, Bates P. 1995. Analysis of the subgroup A avian sarcoma and

leukosis virus receptor: the 40-residue, cysteine-rich, low-density lipoprotein receptor repeat motif of Tva is sufficient to mediate viral entry [J]. J Virol, 69: 4847-4853.

Rubinson DA, Dillon CP, Kwiatkowski A

V, Sievers C, Yang L, Kopinja

J, Rooney DL, Zhang M, Ihriq MM, McManus MT, Gertler FB, Scott ML & van Parijs L. 2003. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference [J]. Nat Genet, 33: 401-406.

Sato M. 2005. Transgenesis via sperm [J]. J Mamm Ova Res, 22:

92-100.

Sato M, Gotoh K, Kimura M. 1999. Sperm-mediated gene transfer by

direct injection of foreign DNA into mouse testis [J]. Transgenics, 2: 357-369.

Sato M, Nakamura S. 2004. A novel gene transmission pattern of

exogenous DNA in offspring obtained after testis-mediated gene transfer (TMGT) [J]. Transgenics, 4: 121-135.

Schaefer-Klein J, Givol I, Barsov EV, Whitcomb JM, VanBrocklin M,

Foster DN, Federspiel MJ, Hughes SH. 1998. The EV-O-derived cell line DF-1 supports the efficient replication of avian leukosis-sarcoma viruses and vectors [J]. Virology, 248: 305-311. Snitkovsky S, Niederman TM, Carter BS, Mulligan RC, Young JAT.

2000. A TVA-single-chain antibody fusion protein mediates specific targeting of a subgroup A avian leukosis virus vector to cells expressing a tumor-specific form of epidermal growth factor receptor [J]. J Virol, 74: 9540-9545.

Snitkovsky S, Niederman TM, Mulligan RC, Young JAT. 2001.

Targeting avian leukosis virus subgroup A vectors by using a TVA-VEGF bridge protein [J]. J Virol, 75: 1571-1575.

Snitkovsky S, Young JAT. 1998. Cell-specific viral targeting mediated

by a soluble retroviral receptor-ligand fusion protein [J]. Proc Natl Acad Sci USA, 95: 7063-7068.

Snitkovsky S, Young JAT. 2002. Targeting retroviral vector infection to

cells that express heregulin receptors using a TVA-heregulin bridge protein [J]. Virology, 292: 150-155.

Soneoka Y, Cannon PM, Ramsdale EE, Griffiths JC, Romano G,

Kingsman SM, Kingsman AJ. 1995. A transient three-plasmid expression system for the production of high titer retroviral vectors [J]. Nucl Acids Res, 23: 628-633.

Warrenfeltz S, Pavlik S, Datta S, Kraemer ET, Benigno B, McDonald

JF. 2004. Gene expression profiling of epithelial ovarian tumors correlated with malignant potential [J]. Mol Cancer, 3: 27-45.

Wills JW, Craven RC, Achacoso JA. 1989. Creation and expression of

myristylated forms of Rous sarcoma virus gag protein in mammalian cells [J]. J Virol, 63: 4331-4343.

Xing D, Orsulic S. 2005. A genetically defined mouse ovarian

carcinoma model for the molecular characterization of pathway-targeted therapy and tumor resistance [J]. Proc Natl Acad Sci USA, 102: 6936-6941.

Yamashita T, Ninomiya M, Acosta PH, García-Verdugo JM, Sunabori T,

Sakaguchi M, Adachi K, Kojima T, Hirota Y, Kawase T, Araki N, Abe K, Okano H, Sawamoto K. 2006. Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum [J]. J Neurosci, 26: 6627-6636.

Yang SY, Wang JG, Cui HX, Sun SG, Li Q, Gu L, Hong Y, Liu PP, Liu

WQ. 2007. Efficient generation of transgenic mice by direct intraovarian injection of plasmid DNA [J]. Biochem Biophys Res Commun, 358: 266-271.

Young JA, Bates P, Varmus HE. 1993. Isolation of a chicken gene that

confers susceptibility to infection by subgroup A avian leukosis and sarcoma viruses [J]. J Virol, 67: 1811-1816..